Optimized FC variants

ABSTRACT

A variant of a parent polypeptide including an Fc region, which variant exhibits increased binding to FcRn as compared to the parent polypeptide and includes at least one amino acid modification in the Fc region.

FIELD OF THE INVENTION

The present invention relates to a variant of a parent polypeptidecomprising an Fc region. The said variant exhibits increased binding toFcRn as compared to the parent polypeptide and comprises at least oneamino acid modification in its Fc region.

DESCRIPTION OF RELATED ART

Monoclonal antibodies are used as therapeutics, to treat a variety ofconditions including cancer, autoimmune diseases, chronic inflammatorydiseases, transplant rejection, infectious diseases, and cardiovasculardiseases. Currently, they are over twenty monoclonal antibodies ormonoclonal antibody fragment products approved on the market, and morethan four hundred in clinical development. Despite such acceptance andpromise, there remains significant need for optimization of thestructural and functional properties of antibodies.

One of the critical issues in the use of monoclonal antibodies intherapy is their persistence in the blood circulation. The rate ofantibody clearance directly affects the efficacy of therapy, andconsequently, the frequency and the quantity of drug administration thatmay cause adverse effects in the patient and also increase medicalcosts.

IgG is the most prevalent immunoglobulin class in humans and also themost utilized in therapeutic. The mechanism of IgG homeostasis has beenelucidated through studies related to the transfer of passive immunityfrom mother to fetus or neonate in rodents (Brambell, 1966, Lancet; 2(7473):1087-93; Rodewald, 1976, J Cell Biol.; 71 (2):666-9; Jones etal., 1972, J Clin Invest., 51 (11):2916-27). In early studies, Brambellhad postulated that there was a receptor for the maternofetaltransmission of IgG and that the mechanism involved in maternofetaltransfer of IgG and catabolism of IgG may be either the same or, atleast, very closely related (Brambell, 1966, Lancet; 2 (7473):1087-93).

Studies have found that the transport of IgG within and across polarizedcells is mediated by binding of Fc region to a high-affinityFc-receptor, named neonatal Fc receptor (FcRn). The FcRn is aheterodimer that comprises a transmembrane α-chain with structuralhomology to the extracellular domains of the α-chain of majorhistocompatibility complex class I molecules, and a soluble light chainconsisting of β₂-microglobulin (β₂m) (Simister and Mostov, 1989, ColdSpring Harb Symp Quant Biol.:54 Pt 1:571-80). In humans, the FcRn isexpressed in placental cells, in intestinal, kidney and bronchialepithelial cells, in endothelial cells and in hematopoetic cells such assmall intestinal macrophages, monocytes and monocyte-derived dendriticcells (Zhu X et al., 2001, J Immunol.; 166:3266-76). FcRn binds its twomajor ligands, IgG and serum albumin, in a pH-dependent manner, withefficient binding at pH 6.0-6.5 and releasing at pH 7.0-7.5 (Raghavan etal., 1995, Biochemistry., 34:14649-57).

The mechanism proposed for IgG protection from catabolism is that IgGsare internalized by non-specific pinocytosis into the endosomes of theendothelial cells where the low pH promotes binding to FcRn (Ghetie andWard, 1997, Nat. Biotechnol., 15: 637-40). Bound IgG-FcRn complexes arerecycled back to the cell surface and dissociate at the neutral pH ofthe extracellular fluid, returning to circulation in the blood. IgGsthat do not bind to FcRn traffic into the lysosomes where they aredegraded by proteases. According to the concentration-dependentcatabolism mechanism for the survival of IgG, at low serum IgGconcentrations the receptor would bind all endocytosed IgG, andefficiently return it to the circulation, yielding a long IgG half-life.Conversely, at high IgG concentrations, the receptor is saturated by IgGand a major fraction of the IgG is unbound by the receptor and trafficsto be degraded, yielding a more rapid catabolism of the unbound IgG.

Various site-specific mutagenesis experiments in the Fc region of mouseIgGs have led to identification of certain critical amino acid residuesinvolved in the interaction between IgG and FcRn (Kim et al., 1994, EurJ Immunol.; 24:2429-34; Kim et al., 1994, Eur J Immunol; 24:542-8;Medesan et al., 1996, Eur J Immunol.; 26:2533-6; Medesan et al., 1997, JImmunol.; 158: 2211-7). These studies and sequence comparison studiesfound that isoleucine at position 253, histidine at position 310, andhistidine at position 435 (according to Kabat numbering, Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)), arehighly conserved in human and rodent IgGs, suggesting their importancein IgG-FcRn binding. These amino acid residues are located at theCH2-CH3 domains interface and the mapping of the functional site tothese residues is consistent with the X-ray crystallographic structureof rat FcRn complexed with rat Fc (Burmeister et al., 1994, Nature; 372(6504):379-83).

Ghetie et al. (1997, Nat. Biotechnol; 15:637-40) randomly mutagenizedposition 252, position 254, and position 256 in a mouse IgG1 Fc-hingefragment. One mutant showed an affinity three and a half times higherfor mouse FcRn and a half-life about 23% or 65% longer in two mousestrains, respectively, as compared to that of the wild-type.

Kim et al. (1999, Eur J Immunol; 29:2819-25) mutagenized human IgG1 byamino acid substitutions at position 253, position 310, or position 435of the Fc region. They found that the mutant Fc-hinge fragments havereduced serum half-lives in mice compared to the wild-type IgG1 Fc-hingefragment, and concluded that Ile253, His310, and His435 play a centralrole in regulating the serum half-life of IgG.

Hornick et al. (2000, J Nucl Med., 41:355-62) showed that a single aminoacid substitution at position 253 in the Fc region of a chimeric humanIgG1 antibody accelerates clearance in mice and improvesimmunoscintigraphy of solid tumors.

Shields et al. (2001, J Biol Chem; 276:6591-604) used alanine scanningmutagenesis to alter residues in the Fc region of a human IgG1 antibodyand then assessed the binding to human FcRn. Positions that effectivelyabrogated binding to FcRn when changed to alanine include I253, S254,H435, and Y436. Other positions showed a less pronounced reduction inbinding as follows: E233-G236, R255, K288, L309, S415, and H433. Severalamino acid positions exhibited an improvement in FcRn binding whenchanged to alanine; notable among these are P238, T256, E272, V305,T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many otheramino acid positions exhibited a slight improvement (D265, N286, V303,K360, Q362, and A378) or no change (S239, K246, K248, D249, M252, E258,T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285,T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301, N315, E318,K320, K322, S324, K326, A327, P329, P331, E333, K334, T335, S337, K338,K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361, Y373,S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401, K414,R416, Q418, Q419, N421, V422, E430, T437, K439, S440, S442, S444, andK447) in FcRn binding.

The most pronounced additivity was found for combination variants withimproved binding to FcRn. At pH 6.0, the E380A/N434A variant showed over8-fold better binding to FcRn, relative to native IgG1, compared with2-fold for E380A and 3.5-fold for N434A. Adding T307A to this effected a12-fold improvement in binding relative to native IgG1.

Dall'Acqua et al. (2002, J Immunol.; 169:5171-80) described randommutagenesis and screening of human IgG1 hinge-Fc fragment phage displaylibraries against mouse FcRn. They disclosed random mutagenesis ofpositions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428,433, 434, and 436. The major improvements in IgG1-human FcRn complexstability occur in substituting residues located in a band across theFc-FcRn interface (M252, S254, T256, H433, N434, and Y436) and to lesserextend substitutions of residues at the periphery like V308, L309, Q311,G385, Q386, P387, and N389. The variant with the highest affinity tohuman FcRn was obtained by combining the M252Y/S254T/T256E andH433K/N434F/Y436H mutations and exhibited a 57-fold increase in affinityrelative to the wild-type IgG1.

Hinton et al. (2004, J Biol Chem.; 279:6213-6) described two mutations,T250Q and M428L, which increased the binding of human IgG2 to human FcRnby about 3 and 7-fold, respectively. In combination, these two mutationsinduced a 28-fold increased binding capacity of IgG2. Injected to rhesusmonkeys for pharmacokinetics studies, both IgG2 mutants, M428L andT250Q/M428L, showed half-lives about 2-fold longer than the wild-typeantibody

Dall'Acqua et al. (2006, J. Biol. Chem.; 281:23514-24) described ahumanized anti-respiratory syncytial virus IgG1 whose Fc region wasmutated at position 252, 254 and 256 (M252Y/S254T/T256E). Thesemutations increase the binding to human FcRn by about 10-fold at pH 6.0while allowing efficient release at pH 7.4 (Dall'Acqua et al., 2002, JImmunol.; 169:5171-80). The in vivo behaviour of such a mutated humanIgG1 exhibited a nearly 4-fold increase in serum half-life in cynomolgusmonkey as compared to wild-type IgG1.

Additionally, various publications describe methods for obtainingphysiologically active molecules whose half-lives are modified either byintroducing an FcRn-binding polypeptide into the molecules (WO 97/43316;U.S. Pat. No. 5,869,046; U.S. Pat. No. 5,747,035; WO 96/32478; WO91/14438) or by fusing the molecules with antibodies whose FcRn-bindingaffinities are preserved but affinities for other Fc receptors have beengreatly reduced (WO 99/43713) or fusing with FcRn binding domains ofantibodies (WO 00/09560; U.S. Pat. No. 4,703,039).

U.S. Pat. No. 6,165,745 discloses a method of producing an antibody witha decreased biological half-life by introducing a mutation into the DNAsegment encoding the antibody. The mutation includes an amino acidsubstitution at position 253, 310, 311, 433, or 434 of the Fc-hingedomain. The full disclosure of U.S. Pat. No. 6,165,745, as well as thefull disclosure of all other U.S. patent references cited herein, arehereby incorporated by reference.

PCT Publication No. WO 00/42072 discloses a polypeptide comprising avariant Fc region with altered FcRn binding affinity, which polypeptidecomprises an amino acid modification at any one or more of amino acidpositions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305,307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388,400, 413, 415, 424, 433, 434, 435, 436, 439, and 447 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex (Kabat et al., op. cit.).

PCT Publication No. WO 02/060919 A2 discloses a modified IgG comprisingan IgG constant domain comprising one or more amino acid modificationsrelative to a wild-type IgG constant domain, wherein the modified IgGhas an increased half-life compared to the half-life of an IgG havingthe wild-type IgG constant domain, and wherein the one or more aminoacid modifications are at one or more of positions 251, 253, 255,285-290, 308-314, 385-389, and 428-435.

There is still a need in the art for novel optimized Fc variants.

SUMMARY OF THE INVENTION

The present invention provides a variant of a parent polypeptide withoptimized properties. The optimized properties comprise higher bindingproperty to FcRn than the corresponding parent polypeptide. In apreferred embodiment, the said variant of a parent polypeptide comprisesa Fc region, exhibits increased binding to FcRn as compared to the saidparent polypeptide, and comprises at least one amino acid modificationin the Fc region of said parent polypeptide, wherein said modificationis selected from the group consisting of 226, 227, 228, 230, 231, 233,234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270,276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302,303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332,334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359,360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383, 384, 385, 386,387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403,404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426,428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fcregion as compared to said parent polypeptide, wherein the numbering ofthe amino acids in the Fc region is that of the EU index as in Kabat.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the variant of the invention.

In another embodiment, the invention provides an isolated nucleic acidencoding the variant of the invention.

In another embodiment, the invention provides a vector comprising thenucleic acid described above.

In another embodiment, the invention provides a host cell containing avector described above.

In another embodiment, the invention provides a method for producing apolypeptide variant comprising culturing the host cell described aboveso that the nucleic acid is expressed.

In another embodiment, the invention provides a medicament comprising avariant of the invention.

In another embodiment, the invention provides the use of a variant ofthe invention for the manufacture of a medicament.

In another embodiment, the invention provides a method for identifyingFc optimized variants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the phagemid vector pMG58 in which human Fc gene encodingamino acid residues 226-447 (EU index as in Kabat) derived from a humanIgG1 heavy chain (Fc226, SEQ no 1) was cloned into.

CVDE: C-terminal part of the VMA1-derived endonuclease, VMA: vacuolarATPase subunit (VMA), CPIII: C-terminal part of the capsid protein pIII(or p3) of the phage M13

FIG. 2 shows the methods used for Fc variants selection in solid phase(2A) and in solution (2B). FcRn-biot refers to biotinylated FcRn andFcRn-p3 refers to FcRn-p3 fusion protein. The Fc-phage is bacteriophageM13 which expresses an Fc variant on its capsid. In solid phaseselection, the wells of immunoplates are coated with FcRn-p3 fusionprotein (Fc-Rn-p3) or with neutravidin followed by FcRn-biot.

FIG. 3 shows the principle of phage-ELISA assay performed on selected Fcvariants. The Fc-phage is a bacteriophage M13 which expresses an Fcvariant on its capsid. FcRn-p3 is FcRn-p3 fusion protein coated on wellsof immunoplates. Anti-M13 refers to mouse anti-M13 antibody fused toHorseradish peroxidase (HRP) used for ELISA detection.

FIG. 4 shows a histogram which represents for each amino acid positionof Fc IgG1 the percentage of mutants comprising a modification at saidposition. X-coordinate: amino acid number according to EU index as inKabat of the mutated position. Y-coordinate: percentage of Fc variantscontaining the position mutated.

FIG. 5a shows the principle of ELISA assay dedicated to measure thebinding affinity of Fc variants for FcRn. FcRn-p3 is an FcRn-p3 fusionprotein coated on wells of immunoplates. Fc is an Fc variant comprisingV5 tag for ELISA detection. Anti-V5 is an anti-V5 antibody fused to HRP.The antibody is used for ELISA detection.

FIG. 5b shows the dose-effect curve for wild-type Fc (rounds) and Fc-Hvariant (squares) obtained by ELISA assay performed as described inExample 1 in IV.1.a. X-coordinate: Concentration of Fc polypeptide.Y-coordinate: percentage of FcRn bound to Fc polypeptide.

FIG. 5c shows the dose-effect curves for wild-type Fc (rounds) andS3A_07 variant (squares) obtained by ELISA assay performed as describedin Example 1 in IV.1.a. X-coordinate: Concentration of Fc polypeptide.Y-coordinate: percentage of FcRn bound to Fc polypeptide.

FIG. 5d shows the dose-effect curves for wild-type Fc (rounds) andS5A_41 variant (squares) obtained by ELISA assay performed as describedin Example 1 in IV.1.a. X-coordinate: Concentration of Fc polypeptide.Y-coordinate: percentage of FcRn bound to Fc polypeptide.

FIG. 6 shows alignments of native human IgG1 sequences referring topositions 216-447 (according to EU index in Kabat) with thecorresponding sequences of human IgG2 (SEQ ID NO:14), human IgG3 (SEQ IDNO:15) and human IgG4 (SEQ ID NO:16). The IgG1 sequences refer to G1m1,17 allotype (SEQ ID NO:12) and to G1 m3 allotype (SEQ ID NO:13). The“lower hinge-CH2-CH3” domain of IgG1 begins at position 216 (see arrow).

FIG. 7 shows the results of ELISA assays which were performed to showthe Fc-variant binding affinity to FcRn at distinct pHs (see for moredetails Example 2, part IV.2). The histogram represents for eachvariants the value of OD_(450nm) measured for ELISA assay performed atpH−6 (black bars), at pH=6.5 (white bars) or at pH=7.4 (grey bars). Thevalue of OD_(450nm) correlates with the amount of immobilized FcRn boundto Fc variants.

FIG. 8a illustrates a schematic map of the expression vector that issued for expressing recombinant IgG1 antibodies bearing Fc variants asdescribed herein. The resulting recombinant IgG1 antibodies possessbinding specificity for the CD20 antigen. As shown in FIG. 8a, thenucleic acid encoding the heavy chain constant region bearing themutations described in the specification and in the examples areinserted between the Apa1 and the Asc1 cloning sites present in theHKCD20-Opti-GA vector.

FIGS. 8b and 8c show SDS-PAGE of IgG variants under non reducingconditions and reducing conditions, respectively.

(1) refers to IgG comprising wild-type Fc; (2) refers to IgG comprisingFc-H variant; (3) refers to IgG comprising C6A_69 variant; (4) refers toIgG comprising C6A_78 variant; (5) refers to IgG comprising T5A_74variant; (6) refers to IgG comprising C6A_74 variant, (7) refers to IgGcomprising C6A_60 variant and (8) refers to comprising C6A_66.

FIG. 9 shows the dose-effect curve for IgG variants of the invention(“1”) and wild-type IgG (“2”) obtained by ELISA assay performed asdescribed in Example 2, III.1 for characterizing the binding of IgGvariants to FcRn. X-coordinate: Concentration of IgG. Y-coordinate:percentage of FcRn bound to IgG.

FIG. 10 shows the dose-effect curves obtained by ELISA assay for IgGvariants of the invention in order to characterize their affinity toFcγRIIIa. (1) refers to the curve obtained for C6A_66 variant, (2)refers to the curve obtained for Rituximab and (3) refers to curvesobtained for C6A_69; C6A_78; T5A_74; C6A_74; C6A_60 variants, andwild-type IgG. The ELISA assay was performed as described in Example 2,in part IV.1.a. X-coordinate: Concentration of IgG. Y-coordinate:percentage of FcγRIIIa bound to IgG.

FIG. 11 illustrates the binding of various recombinant IgG to JurkatFcRn. FIG. 11 shows the binding or Ritixan and of various variantsaccording to the invention to Jurkat FcRn has been determined asdescribed in the Materials and Methods Section above and expressed asmean fluorescence intensity (MFI) values.

FIG. 12 shows the dose-effect curves obtained in ADCC assay for de IgGvariants of the invention. (1) refers to the curve of C6A_66 variant,(2) refers to the curve of Rituximab and (3) refers to the curves ofLFB-R603, WT-IgG, and IgG variants of the invention (namely C6A_69;C6A_78; T5A_74; C6A_74; C6A_60 variants). X-coordinate: Concentration ofIgG. Y-coordinate: percentage of cell lysis.

DETAILED DESCRIPTION OF THE INVENTION

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

Throughout the present specification and claims, the numbering of theresidues in the Fc region is that of the immunoglobulin heavy chainaccording to the EU index as in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), expressly incorporatedherein by reference. The “EU index as in Kabat” refers to the residuenumbering of the human IgG1 EU antibody.

By “polypeptide” or “protein” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides.

By “amino acid” as used herein is meant one of the 20 naturallyoccurring amino acids or any non-natural analogues that may be presentat a specific, defined position.

The naturally occurring amino acids can be abbreviated with the threeletter code, or with the one letter code:

Amino acid Three letter code One letter code alanine ala A arginine argR asparagine asn N aspartic acid asp D asparagine or aspartic acid asx Bcysteine cys C glutamic acid glu E glutamine gln Q glutamine or glutamicacid glx Z glycine gly G histidine his H isoleucine ile I leucine leu Llysine lys K methionine met M phenylalanine phe F proline pro P serineser S threonine thr T tryptophan try W tyrosine tyr Y valine val V

By “position” as used herein is meant a location in the sequence of aprotein. For Fc region, the positions are numbered according to the EUindex as in Kabat.

By “amino acid modification” herein is meant a change in the amino acidsequence of a polypeptide. “Amino acid modifications” which may be alsotermed “amino acid changes” herein include amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution N434S refers to avariant polypeptide, in this case an Fc variant, in which the asparagineat position 434 is replaced with serine. By “amino acid insertion” or“insertion” as used herein is meant the addition of an amino acid at aparticular position in a parent polypeptide sequence. For example,insert G>235-236 designates an insertion of glycine between positions235 and 236. By “amino acid deletion” or “deletion” as used herein ismeant the removal of an amino acid at a particular position in a parentpolypeptide sequence. For example, E294del designates the deletion ofglutamic acid at position 294.

For example, the following format of modifications is preferentiallyused: 434S, or N434S, means that the parent amino acid in position 434,i.e. asparagine, is replaced by serine.

In case of a combination of substitutions, the preferred format is thefollowing: 259I/315D/434Y or V259I/N315D/N434Y. That means that thereare three substitutions in the variant, one in positions 259, one inposition 315 and one in position 434, and that amino acid in position259 of the parent polypeptide, i.e. valine, is replaced by isoleucine,that the amino acid in position 315 of the parent polypeptide, i.e.asparagine, is replaced by aspartic acid and that the amino acid inposition 434 of the parent polypeptide, i.e. asparagine, is replaced bytyrosine.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.Variables regions comprise Complementarity-Determining Regions (CDRs)and Framework Regions (FR).

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, the lastthree constant region immunoglobulin domains of IgE and IgM, and theflexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3 which are CH2 and CH3 domains,respectively for IgGs) and the lower hinge region between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). The human IgG1 heavy chain Fc region is definedherein to comprise residues C226 to its carboxyl-terminus, wherein thenumbering is according to the EU index as in Kabat. In the context ofhuman IgG1, the lower hinge refers to positions 226-236, the CH2 domainrefers to positions 237-340 and the CH3 domain refers to positions341-447 according to the EU index as in Kabat. The corresponding Fcregion of other immunoglobulins can be identified by sequencealignments.

Fc may refer to this region in isolation, or this region in the contextof an Fc polypeptide, as described below. By “Fc polypeptide” as usedherein is meant a polypeptide that comprises all or part of an Fcregion. Fc polypeptides include, but are not limited to, antibodies, Fcfusions, isolated Fcs, Fc-conjugates and Fc fragments.

The term “antibody” is used herein in the broadest sense. “Antibody”refers to any polypeptide which at least comprises (i) a Fc region and(ii) a binding polypeptide domain derived from a variable region of animmunoglobulin. The said binding polypeptide domain is able to bindspecifically one given target antigen or a group of target antigens. Abinding polypeptide domain which derives from a variable region of animmunoglobulin comprises one or more CDRs. Antibodies include, but arenot limited to, full-length immunoglobulins, monoclonal antibodies,multi-specific antibodies, Fc-fusion protein comprising at least onevariable region, synthetic antibodies (sometimes referred to herein as“antibody mimetics”), chimeric antibodies, humanized antibodies, fullyhuman antibodies, antibody-fusion proteins, antibody conjugates andfragments of each respectively.

By “full-length antibody” or by “immunoglobulin” as used herein is meantthe structure that constitutes the natural biological form of anantibody, including variable and constant regions. “Full lengthantibody” covers monoclonal full-length antibodies, wild-typefull-length antibodies, chimeric full-length antibodies, humanizedfull-length antibodies, the list not being limitative.

In most mammals, including humans and mice, the structure of full-lengthantibodies is generally a tetramer. Said tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(typically having a molecular weight of about 25 kDa) and one “heavy”chain (typically having a molecular weight of about 50-70 kDa). In somemammals, for example in camels and llamas, full-length antibodies mayconsist of only two heavy chains, each heavy chain comprising a variabledomain attached to the Fc region.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. In the variable region, three loops are gathered for eachof the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see Sequences of Immunological Interest, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., incorporatedby reference herein in its entirety).

In the case of human immunoglobulins, light chains are classified askappa and lambda light chains. Heavy chains are classified as mu, delta,gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subclasses, including,but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses,including, but not limited to, IgM1 and IgM2. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Theknown human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, IgM1, IgM2, IgD, and IgE.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans, IgG comprises the subclasses or isotypes IgG1,IgG2, IgG3, and IgG4. In mice, IgG comprises IgG1, IgG2a, IgG2b, IgG3.Full-length IgGs are tetramers and consist of two identical pairs of twoimmunoglobulin chains, each pair having one light and one heavy chain,each light chain comprising immunoglobulin domains VL and CL, and eachheavy chain comprising immunoglobulin domains VH, Cγ1 (also called CH1),Cγ2 (also called CH2), and Cγ3 (also called CH3). In the context ofhuman IgG1, “CH1” refers to positions 118-220, CH2 domain refers topositions 237-340 and CH3 domain refers to positions 341-447 accordingto the EU index as in Kabat. IgG heavy chain also comprises a hingedomain which refers to positions 221-236 in the case of IgG1.

By “parent polypeptide” or “polypeptide parent” as used herein is meantan unmodified polypeptide that is subsequently modified to generate avariant. Said parent polypeptide may be a naturally occurringpolypeptide, a variant of a naturally occurring polypeptide, engineeredversion of a naturally occurring polypeptide or a synthetic polypeptide.Parent polypeptide may refer to the polypeptide itself, or the aminoacid sequence that encodes it. In the context of the present invention,the parent polypeptide comprises an Fc region selected from the group ofwild-type Fc regions, their fragments and their mutants. Accordingly,the parent polypeptide may optionally comprise pre-existing amino acidmodifications in its Fc region (i.e. an Fc mutant) as compared towild-type Fc regions.

Advantageously, the parent polypeptide is an antibody, animmunoglobulin, an Fc fusion polypeptide, an Fc conjugate, this list notbeing limitative. Accordingly, by “Parent immunoglobulin” as used hereinis meant immunoglobulin polypeptide that is modified to generate avariant immunoglobulin, and by “parent antibody” as used herein is meantantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes, but are not limited to, knowncommercial, recombinantly produced antibodies.

As used herein, the term “at least one” is equal to “one or more”.

By “variant polypeptide”, “polypeptide variant” or “variant” as usedherein is meant a polypeptide sequence that differs from that of aparent polypeptide sequence by virtue of at least one amino acidmodification.

Variant may refer to Fc variant, Fc polypeptide variant, proteinvariant, antibody variant, immunoglobulin variant, IgG variant, thislist not being limitative.

By “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. The parent polypeptide may be a naturally occurring orwild-type (WT) polypeptide, or may be a modified version of a WTpolypeptide.

Parent polypeptides of interest are polypeptides which comprise an Fcregion as defined above. Preferably the variant of the invention has apolypeptide sequence that differs from that of a parent polypeptidesequence by virtue of at least one amino acid modification in the Fcregion. Consequently a variant of interest comprises an Fc variant.

Accordingly, by “Fc variant” or “variant Fc” as used herein is meant anFc sequence that differs from that of a parent Fc sequence by virtue ofat least one amino acid modification. An Fc variant may be an isolatedFc region and fragments thereof, or may exist in the context of anantibody, Fc fusion, and fragments therefore, the list not beinglimitative.

By “protein variant” or “variant protein” as used herein is meant aprotein that differs from a parent protein by virtue of at least oneamino acid modification. By “antibody variant” or “variant antibody” asused herein is meant an antibody that differs from a parent antibody byvirtue of at least one amino acid modification. By “IgG variant” or“variant IgG” as used herein is meant an antibody that differs from aparent IgG by virtue of at least one amino acid modification.Preferably, the variant has at least one amino acid modificationcompared to the parent polypeptide, e.g. from about 1 to about 45 aminoacid modifications, preferably from about 1 to about 20 amino acidmodifications, and more preferably from about 1 to about 10 amino acidmodifications.

The variant sequence herein will preferably possess at least about 80%identity with its parent polypeptide sequence, and most preferably atleast about 90% identity.

As intended herein, a determined polypeptide having at least about 90%amino acid identity with a reference polypeptide possesses at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% aminoacid identity with the said reference polypeptide.

To determine the percent of identity of two amino acid sequences, thesequences are aligned for optimal comparison purposes. For example, gapscan be introduced in one or both of a first and a second amino acidsequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes. For optimal comparison purposes,the percent of identity of two amino acid sequences can be achieved withCLUSTAL W (version 1.82) with the following parameters: (1) CPUMODE=ClustalW mp; (2) ALIGNMENT=<<full<<; (3) OUTPUT FORMAT=<<alnw/numbers<<; (4) OUTPUT ORDER=<<aligned<<; (5) COLOR ALIGNMENT=<<no<<;(6) KTUP (word size)=<<default<<; (7) WINDOW LENGTH=<<default<<; (8)SCORE TYPE=<<percent<<; (9) TOPDIAG=<<default<<; (10)PAIRGAP=<<default>>; (11) PHYLOGENETIC TREE/TREE TYPE=>>none>>; (12)MATRIX=>>default>>; (13) GAP OPEN=>>default>>; (14) ENDGAPS=>>default>>; (15) GAP EXTENSION=>>default>>; (16) GAP DISTANCES=>>,default>>; (17) TREE TYPE=>>cladogram>> et (18) TREE GRAPDISTANCES=>>hide>>.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc. have an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FCRN gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FCRN gene.Unless otherwise noted herein, FcRn or FcRn protein refers to thecomplex of α-chain with beta-2-microglobulin. In human, the gene codingfor FcRn is called FCGRT.

By “increased FcRn binding” as used herein is meant the increase inbinding affinity, in vivo or in vitro, of the variant of the inventionto FcRn, compared to the parent polypeptide. The ability of thepolypeptide variant to bind an FcRn may be evaluated in vitro by ELISA(Example 1 part IV.1.a) or SPR technology (Example 1 part IV.1.b.). Thevariants which have an enhanced binding property for FcRn most oftenhave an enhanced serum retention in vivo and, thus, an increasedhalf-life.

In order to increase the retention of the Fc region in vivo, theincrease in binding affinity for FcRn must occur at around pH 6, whilemaintaining lower affinity at around pH 7.4.

Although still under examination, Fc regions are believed to have alonger half-life in vivo, because the binding to FcRn at pH 6 allow thesequestration of Fc regions into endosomes (Ghetie and Ward, 1997Immunol Today. 18 (12): 592-598, incorporated by reference herein in itsentirety). The endosomal compartment then recycles the Fc regions to thecell surface. Once the compartment opens to the extracellular space, thehigher pH, almost 7.4, induces the release of Fc regions back into theblood. Therefore, the amino acid modifications in the Fc region thatwill increase Fc regions' half-life in vivo will ideally increase FcRnbinding at the lower pH while still allowing release of Fc region athigher pH.

The term “in vivo half-life” as used herein refers to a biologicalhalf-life of a polypeptide of interest in the circulation of a givenanimal and is represented by the time required for half the quantitypresent in the circulation of the animal to be cleared from thecirculation and/or other tissues in the animal.

The present invention is based on the identification of amino acidmodifications of Fc region which modifications increase the bindingaffinity of the Fc region for FcRn. The amino acid modifications ofinterest have been determined by generating two Fc variants libraries byrandom mutagenesis and by measuring the binding property of saidvariants for FcRn.

Accordingly, the present invention relates to variants of parentpolypeptides comprising an Fc region which display increased binding toFcRn as compared to said parent polypeptides.

A parent polypeptide of the invention is a polypeptide comprising an Fcregion. Said polypeptide may comprise one single polypeptide chain orseveral polypeptide chains which are not covalently linked together.Parent polypeptides include, but are not limited to, antibodies, Fcfusion proteins, Fc conjugates, Fc derivated polypeptides, isolated Fcand fragments thereof. As a consequence, said parent polypeptide may bea naturally occurring polypeptide, a variant of a naturally occurringpolypeptide, an engineered version of a naturally occurring polypeptide,a synthetic polypeptide or a polypeptide comprising a non-proteinousfragment. An engineered version of a naturally occurring polypeptide isa polypeptide with is not encoded by a naturally occurring gene. Forexample, the engineered polypeptide may be a chimeric antibody or ahumanized antibody.

The Fc region of the parent polypeptide is preferably selected from thegroup consisting of wild-type Fc regions of IgGs, fragments and mutantsthereof. Herein, Fc region of IgG corresponds to the “lower hinge”-CH2-CH3 domain (For IgGs, CH2 and CH3 are also called Cγ2 and Cγ3domains). The sequence of “lower hinge” -CH2-CH3 domain of the wild typehuman IgG1 is the sequence of SEQ ID NO:1. In the context of human IgG1,the lower hinge refers to positions 226-236, the CH2 domain refers topositions 237-340 and the CH3 domain refers to positions 341-447according to the EU index as in Kabat. The analogous domains for otherIgG sub-classes can be determined from amino acid sequence alignment ofheavy chains or heavy chain fragments of said IgG sub-classes with thatof human IgG1.

Fragments of Fc region are defined as polypeptides which comprise one ormore polypeptides derived from a wild-type Fc region, preferably fromthe “lower hinge-CH2-CH3” domain of a wild-type IgG. The said fragmentshave a dissociation constant for FcRn lower than 1 microM according tothe SPR assay described in Example 1 part IV.1.).

As mentioned above, the parent polypeptide can comprise a wild-type Fcmutant i.e a Fc region which already comprises pre-existing amino acidmodifications such as additions, insertions and/or substitutions withproviso that the said Fc mutant has a dissociation constant for FcRnlower than 1 microM according to the SPR assay described in Example 1part IV.1. and is not a wild-type Fc region.

By “variant polypeptide” or “variant” as used herein is meant apolypeptide sequence which differs from that of a parent polypeptide invirtue of at least one amino acid modification.

The variant polypeptide according to the present invention displays anincreased binding to FcRn as compared to the corresponding parentpolypeptide. In other words, the affinity of the variant for FcRn ishigher than that of the parent polypeptide. Such variants are optimizedvariants according to the invention.

The affinity of the said polypeptides for FcRn can be evaluated bywell-known methods of the prior art. For example, the one skilled in theart may determine the dissociation constant (Kd) using Surface PlasmonResonance (SPR) experiments as illustrated in the Example 1 part IV.1.b.of the present application. If the variant has a Kd 1.1-fold lower thanthat of its corresponding parent then the said variant is an optimizedvariant according to the invention.

As an alternative, the one skilled in the art may perform an appropriateELISA assay. An appropriate ELISA assay enables to compare the bondstrength of the variant and that of the parent to FcRn as illustrated inExample 1. The specific signals detected for the variant and the parentpolypeptide are compared. The variant is an optimized variant of theinvention if its specific signal is at least 1.2-fold stronger, morepreferably at least 3.2-fold stronger than that of the parentpolypeptide (i.e. at least as good as the Fc variant having the doubleamino acid modification T250Q/M428L).

Appropriate ELISA assays are illustrated in Example 1 of the presentapplication. The binding affinity can be indifferently determined byevaluating the full-length polypeptides (see Example 2 part III) or byevaluating the isolated Fc regions thereof (see Example 1 part IV).

According to the invention, polypeptide variants of interest comprise atleast one amino acid modification in its Fc region as compared to theparent polypeptide. The amino acid modifications are selected from thegroup consisting of amino acid insertions, deletions and substitutions.

The applicants have shown that in order to obtain a polypeptide varianthaving increased binding to FcRn as compared to its parent polypeptide,the at least one amino acid modification should be introduced at anamino acid position selected from the group consisting of 226, 227, 228,230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265,267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298,299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325,327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354,355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383,384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421,422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447of the Fc region as compared to said parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat.

Herein, the “EU index as in Kabat” refers to the residue numbering ofthe human IgG1 EU antibody. For example, the analogous positions forother Fc regions can be determined from amino acid sequence alignment ofthe said Fc regions with human IgG1 heavy chain fragment comprising thepolypeptide of SEQ ID NO:1. For illustrative purpose, FIG. 6 depicts thesequence alignment of human IgG1, IgG2, IgG3 and IgG4 heavy chainfragments comprising “lower hinge-CH2-CH3” domain.

By “at least one amino acid modification” as used herein means “one ormore modifications”. It is considered that the introduction of more than20 amino acid modifications in the Fc region may drastically impair itsbiological activities. Accordingly, the polypeptide variant preferablyhas from 1 to 20 and more preferably from 1 to 10 amino acidmodifications, at positions selected from the list cited above. By “1 to20 amino acid modifications” as used herein encompasses 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acidmodifications. The said polypeptide variant sequence preferablypossesses at least about 90% identity with its parent polypeptidesequence.

As intended herein, a determined polypeptide having at least about 90%amino acids with a reference polypeptide possesses at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% amino acidsidentity with said reference polypeptide.

The Fc variants of the present invention which display the highestbinding affinity for FcRn generally comprise more than one amino acidmodifications. The results obtained from phage ELISA assay described inexample II shows that the optimized variants comprising more than oneamino acid modification may have a specific signal from about 3.2-foldto 30-fold stronger (see table 2 and table 3) than the wild-type Fcwhereas the variants with a single point amino acid modification (seetable 1) may have a signal from about 1.2-fold to 3.5-fold stronger thanthe wild-type Fc. As illustrated in table 3, the signal of the optimizedvariant may be from about 1-fold to about 10-fold stronger than that ofFc-H which refers to the Fc variant having the double amino acidmodification T250Q/M428L.

Accordingly, in a specific embodiment, the said variant comprises atleast two amino acid modifications selected from the list consisting of226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256,259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292,294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317,320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347,350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378,380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418,419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444,445, 446 and 447 of the Fc region as compared to said parentpolypeptide, wherein the numbering of the amino acids in the Fc regionis that of the EU index as in Kabat.

As described in table 5 of the present application, Fc variants whichdisplay the highest binding affinity for FcRn may have 3 to 6 amino acidmodifications.

Accordingly, in a further embodiment, the variant polypeptides of theinvention may comprise 3 to 6 amino acid modifications at amino acidpositions selected from the group consisting of 226, 227, 228, 230, 231,233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269,270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301,302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330,332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356,359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 383, 384, 385,386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424,426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fcregion as compared to said parent polypeptide, wherein the numbering ofthe amino acids in the Fc region is that of the EU index as in Kabat.

The amino acid modifications are preferably selected from the group ofdeletions and substitutions.

Some amino acid positions of the above list—namely 226, 230, 241, 264,307, 315, 330, 342, 362, 378, 382, 389, 396, 397, 421 and 434—are keypositions. In other words, the Fc variants which display high bindingaffinity for FcRn are likely to comprise at least one amino acidmodification at the said amino acid positions.

In certain embodiments, the polypeptide variant according to theinvention comprises at least one amino acid modification at amino acidpositions selected from the group consisting of 226, 230, 241, 264, 307,315, 330, 342, 362, 378, 382, 389, 396, 397, 421 and 434 of the Fcregion as compared to the parent polypeptide, wherein the numbering ofthe amino acids in the Fc region is that of the EU index as in Kabat.

Among the above key positions, the sequencing of the Fc variants whichdisplay the strongest binding for FcRn have shown that the amino acidpositions 230, 264, 307, 315, 330, 378 and 434 are the most oftenmutated positions. Accordingly, in another embodiment, the at least onemodification occurs at one position selected from the group consistingof 230, 264, 307, 315, 330, 378 and 434, more preferably from the groupconsisting of 264, 315, 378 and 434 of the Fc region as compared to theparent polypeptide, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat.

As mentioned above, the introduction of at least two amino acidmodifications can noticeably enhance the binding affinity of Fc variantfor FcRn as compared to the Fc parent.

Accordingly, in an alternate embodiment, the polypeptide variantcomprises at least two amino acid modifications, said at least two aminoacid modifications comprising:

-   -   (i) one modification at an amino acid position selected from the        group consisting of 226, 230, 241, 264, 307, 315, 330, 342, 362,        378, 382, 389, 396, 397, 421, and 434; and    -   (ii) at least one modification at an amino acid position        selected from the group consisting of 226, 227, 228, 230, 231,        233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267,        269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298,        299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322,        325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,        352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378,        380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395,        396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415,        416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439,        440, 443, 444, 445, 446 and 447,

of the Fc region as compared to the parent polypeptide wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modification (i) does not occurat the same amino acid position as the modification (ii).

For example, according to the said proviso, if the amino acidmodification (i) occurs at position 434, the at least one amino acidmodification (ii) can occur at any position of the list cited in (ii)except on position 434.

In another embodiment, the polypeptide variant comprises at least twoamino acid modifications, said at least two amino acid modificationscomprising:

-   -   (i) one modification at an amino acid position selected from the        group consisting of 264, 315, 378 and 434; and    -   (ii) at least one modification at an amino acid position        selected from the group consisting of 226, 227, 228, 230, 231,        233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267,        269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298,        299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322,        325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,        352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378,        380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395,        396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415,        416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439,        440, 443, 444, 445, 446 and 447

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso that themodification (i) does not occur at the same amino acid position as themodification (ii).

In an additional embodiment, the said variant comprises at least twoamino acid modifications, said at least two amino acid modificationscomprising:

-   -   (i) one amino acid modification at a position selected from the        group consisting of 264, 315, 378 and 434; and    -   (ii) at least one amino acid modification at a position selected        from the group consisting of 226, 230, 241, 264, 307, 315, 330,        342, 362, 378, 382, 389, 396, 397, 421 and 434

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso that themodification (i) does not occur at the same amino acid position as themodification (ii).

In another additional embodiment the said variant comprises at least twoamino acid modifications comprising:

-   -   (i) one amino acid modification at a position selected from the        group consisting of 378 and 434; and    -   (ii) at least one amino acid modification at a position selected        from the group consisting of 226, 230, 241, 264, 307, 315, 330,        342, 362, 378, 382, 389, 396, 397, 421 and 434

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso that themodification (i) does not occur at the same amino acid position as themodification (ii).

In an alternate embodiment, the polypeptide variant comprises at leastthree amino acid modifications in its Fc region. Accordingly, the saidat least three amino acid modifications may comprise:

-   -   (i) two modifications at two amino acid positions selected from        the group consisting of 226, 230, 241, 264, 307, 315, 330, 342,        362, 378, 382, 389, 396, 397, 421 and 434; and    -   (ii) at least one modification at an amino acid position        selected from the group consisting of 226, 227, 228, 230, 231,        233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267,        269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298,        299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322,        325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,        352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378,        380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395,        396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415,        416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439,        440, 443, 444, 445, 446 and 447

of the Fc region as compared to the parent polypeptide wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modification (i) does not occurat the same amino acid position as the modification (ii).

In an alternate embodiment, the polypeptide variant comprises at leastthree amino acid modifications, said at least three amino acidmodifications comprising:

-   -   (i) one modification at an amino acid position selected from the        group consisting of 264, 315, 378 and 434;    -   (ii) one modification at an amino acid position selected from        the group consisting of 226, 230, 241, 264, 307, 315, 330, 342,        362, 378, 382, 389, 396, 397, 421 and 434; and    -   (iii) at least one modification at an amino acid position        selected from the group consisting of 227, 228, 230, 231, 233,        234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269,        270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299,        301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325,        327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352,        354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380,        382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396,        397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416,        418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440,        443, 444, 445, 446 and 447

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso thatmodification (i), modification (ii) and modification (iii) do notsimultaneously occur at the same amino acid positions.

In other embodiments, the polypeptide variant comprises at least threeamino acid modifications, said at least three amino acid modificationscomprising:

-   -   (i) one modification at an amino acid position selected from the        group consisting of 378 and 434;    -   (ii) one modification at an amino acid position selected from        the group of 226, 230, 241, 264, 307, 315, 330, 342, 362, 378,        382, 389, 396, 397, 421 and 434; and    -   (iii) at least one modification at an amino acid position        selected from the group consisting of 227, 228, 230, 231, 233,        234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269,        270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299,        301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325,        327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352,        354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380,        382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396,        397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416,        418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440,        443, 444, 445, 446 and 447

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso thatmodification (i), modification (ii) and modification (iii) do notsimultaneously occur at the same amino acid positions.

In all previously cited embodiments of the present invention, the aminoacid modifications are preferably selected from the group consisting ofamino acid substitutions and deletions.

A further object of the invention relates to a variant of a parentpolypeptide comprising a Fc region which exhibits increased binding toFcRn as compared to said parent polypeptide and comprises at least oneamino acid modification in the Fc region selected from the groupconsisting of 226G, 226Y, 227S, 227L, 228R, 228L, 230S, 230T, 230L,230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L, 241Y, 241R, 243L, 246R,250A, 252L, 256N, 259I, 264A, 264E, 264M, 265G, 265N, 267N, 267R, 269D,269G, 270N, 270E, 276S, 284L, 285Y, 288R, 2891, 290R, 290E, 291S, 291Q,292W, 294del, 297D, 298G, 298N, 299M, 299A, 299K, 301C, 302A, 303A,3031, 305A, 307P, 307A, 307N, 308I, 309P, 311R, 315D, 317R, 320T, 320E,322R, 325S, 327V, 327T, 330V, 330T, 332V, 334E, 334R, 335A, 338R, 340E,342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A, 352S, 354P, 355Q, 355G,356N, 359A, 360N, 360R, 361D, 361S, 362R, 362E, 369A, 370R, 371D, 375A,375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R, 383N, 384I, 384T, 385R,386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S, 392E, 392R, 393N, 394A,395A, 395S, 396S, 396L, 397A, 397M, 398P, 399N, 400P, 401A, 401G, 403T,404L, 408T, 411A, 412A, 414R, 415D, 415N, 416K, 416G, 418R, 418K, 418E,419H, 420R, 421T, 421S, 421D, 422A, 424L, 426T, 428L, 433R, 433P, 434Y,434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F, 444P, 445S, 446A, 447Eand 447N of the Fc region, as compared to the parent polypeptide,wherein the numbering of the amino acids in the Fc region is that of theEU index as in Kabat.

In an alternate embodiment, the said polypeptide comprises at least onemodification selected from the group consisting of 226G, 227L, 230S,230T, 230L, 231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E,294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V, 330V,342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T, 382V, 383N, 386R,386K, 387T, 389T, 389K, 392R, 395A, 396L, 397M, 403T, 404L, 415N, 416K,421T, 426T, 428L, 433R, 434Y, 434S and 439R of the Fc region, ascompared to the parent polypeptide, wherein the numbering of the aminoacids in the Fc region is that of the EU index as in Kabat.

Preferably, the said variant has from 1 to 20, more preferably from 1 to10 amino acid modifications selected from the above lists, as comparedto the parent polypeptide. As used herein, by “from 1 to 20modifications” is meant 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 and 20 modifications.

In some embodiments, the said variant comprises from 3 to 6 amino acidmodifications selected from the group consisting of 226G, 226Y, 227S,227L, 228R, 228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R,239A, 241L, 241Y, 241R, 243L, 246R, 250A, 252L, 256N, 259I, 264A, 264E,264M, 265G, 265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y,288R, 2891, 290R, 290E, 291S, 291Q, 292W, 294del, 297D, 298G, 298N,299M, 299A, 299K, 301C, 302A, 303A, 3031, 305A, 307P, 307A, 307N, 308I,309P, 311R, 315D, 317R, 320T, 320E, 322R, 325S, 327V, 327T, 330V, 330T,332V, 334E, 334R, 335A, 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G,347R, 350A, 352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R, 361D, 361S,362R, 362E, 369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V,382G, 383R, 383N, 384I, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K,389R, 390S, 392E, 392R, 393N, 394A, 395A, 395S, 396S, 396L, 397A, 397M,398P, 399N, 400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A, 414R, 415D,415N, 416K, 416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422A,424L, 426T, 428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N,443R, 444F, 444P, 445S, 446A, 447E and 447N of the Fc region, ascompared to the parent polypeptide, wherein the numbering of the aminoacids in the Fc region is that of the EU index as in Kabat.

In an alternate embodiment, the said polypeptide comprises from 3 to 6amino acid modifications selected from the group consisting of 226G,227L, 230S, 230T, 230L, 231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G,267R, 290E, 294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S,327V, 330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T, 382V,383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L, 397M, 403T, 404L,415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S and 439R of the Fcregion, as compared to the parent polypeptide, wherein the numbering ofthe amino acids in the Fc region is that of the EU index as in Kabat.

Some amino acid modifications of the above lists are key modifications.In other words, the Fc variants which display high binding affinity toFcRn are likely to comprise at least one amino acid modificationselected from the said key modifications.

Accordingly, the said polypeptide variant may comprise at least onemodification selected from the group consisting of 226G, 230S, 230T,230L, 241L, 264E, 307P, 315D, 330V, 342R, 362R, 362E, 378V, 378T, 382V,389T, 389K, 396L, 397M, 421T, 434Y and 434S of the Fc region compared tosaid parent polypeptide, wherein the numbering of the amino acids in theFc region is that of the EU index as in Kabat.

In another embodiment, the said polypeptide variant comprises at leastone amino acid modification selected from the group consisting of 264E,315D, 378V, 378T, 434Y and 434S of the Fc region as compared to saidparent polypeptide, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat.

In a further embodiment, the said polypeptide variant comprises at leastone amino acid modification selected from the group consisting of 378V,378T, 434Y and 434S of the Fc region as compared to said parentpolypeptide, wherein the numbering of the amino acids in the Fc regionis that of the EU index as in Kabat.

As mentioned above, the introduction of at least two amino acidmodifications can noticeably enhance the binding of Fc variants to FcRnas compared to the parents. At least one of said modifications may beselected from the key modifications i.e. from the group consisting of226G, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 342R, 362R, 362E,378V, 378T, 382V, 389T, 389K, 396L, 397M, 421T, 434Y and 434S.

In an alternate embodiment, the said polypeptide variant comprises atleast two amino acid modifications, the said at least two modificationscomprising

-   -   (i) one modification selected from the group consisting of 226G,        230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 342R, 362R,        362E, 378V, 378T, 382V, 389T, 389K, 396L, 397M, 421T, 434Y and        434S; and    -   (ii) at least one amino acid modification at an amino acid        position selected from the group consisting of 227, 228, 230,        231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265,        267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297,        298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320,        322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347,        350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375,        378, 380, 382, 383, 384, 385, 386, 387, 389, 390, 392, 393, 394,        395, 396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414,        415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438,        439, 440, 443, 444, 445, 446 and 447

of the Fc region, as compared to the parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modification (i) does not occurat the same amino acid position as the modification (ii).

In a further embodiment, the said variant comprises at least two aminoacid modifications, the said at least two modifications comprising

-   -   (i) one amino acid modification selected from the group        consisting of 378V, 378T, 434Y and 434S; and    -   (ii) at least one amino acid modification at an amino acid        position selected from the group consisting of 226, 230, 241,        264, 307, 315, 330, 342, 362, 378, 382, 389, 396, 397, 421 and        434

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso that themodification (i) does not occur at the same amino acid position as themodification (ii).

In another embodiment, the said variant comprises at least two aminoacid modifications, the said at least two modifications comprising:

-   -   (i) one amino acid modification selected from 378V, 378T, 434Y        and 434S; and    -   (ii) at least one amino acid modification selected from 226G,        230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 342R, 362R,        362E, 378V, 378T, 382V, 389T, 389K, 396L, 397M, 421T, 434Y and        434S, and more preferably, from 226G, 230S, 230T, 230L, 241L,        264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K, 434Y and        434S

of the Fc region, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat and with the proviso that themodification (i) does not occur at the same amino acid position as themodification (ii).

Accordingly, a further object of the invention relates to a variant of aparent polypeptide comprising an Fc region which exhibits increasedbinding to FcRn as compared to said parent polypeptide and comprises atleast one combination of amino acid modifications in the Fc region.

The at least one combination of modifications is selected from the groupconsisting of: 226G/330V, 230L/264E, 230L/378V, 230S/315D, 230S/434Y,230T/378V, 241L/434S, 250A/434Y, 264E/378T, 305A/315D, 305A/330V,305A/434Y, 307P/434Y, 315D/389T, 330V/382V, 330V/389T, 378V/421T,389K/434Y, 389T/434Y, 396L/434S, 230T/264E, 230T/315D, 230T/434S,230T/434Y, 241L/307P, 264E/307P, 264E/396L, 315D/362R, 315D/382V,362R/434Y, 378V/434Y, 382V/434Y, 226G/315D, 226G/434Y, 241L/378V,307P/378V, 241L/264E, 378V/434S, 264E/378V, 264E/434S, 315D/330V,330V/434Y and 315D/434Y of the Fc region, wherein the numbering of theamino acids in the Fc region is that of the EU index as in Kabat.

The said variant may further comprise at least one modification selectedfrom the group of 226G, 226Y, 227S, 227L, 228R, 228L, 230S, 230T, 230L,230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L, 241Y, 241R, 243L, 246R,250A, 252L, 256N, 259I, 264A, 264E, 264M, 265G, 265N, 267N, 267R, 269D,269G, 270N, 270E, 276S, 284L, 285Y, 288R, 2891, 290R, 290E, 291S, 291Q,292W, 294del, 297D, 298G, 298N, 299M, 299A, 299K, 301C, 302A, 303A,3031, 305A, 307P, 307A, 307N, 308I, 309P, 311R, 315D, 317R, 320T, 320E,322R, 325S, 327V, 327T, 330V, 330T, 332V, 334E, 334R, 335A, 338R, 340E,342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A, 352S, 354P, 355Q, 355G,356N, 359A, 360N, 360R, 361D, 361S, 362R, 362E, 369A, 370R, 371D, 375A,375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R, 383N, 384I, 384T, 385R,386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S, 392E, 392R, 393N, 394A,395A, 395S, 396S, 396L, 397A, 397M, 398P, 399N, 400P, 401A, 401G, 403T,404L, 408T, 411A, 412A, 414R, 415D, 415N, 416K, 416G, 418R, 418K, 418E,419H, 420R, 421T, 421S, 421D, 422A, 424L, 426T, 428L, 433R, 433P, 434Y,434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F, 444P, 445S, 446A, 447Eand 447N of the Fc region, as compared to the parent polypeptide,wherein the numbering of the amino acids in the Fc region is that of theEU index as in Kabat.

In another embodiment, a variant according to the present inventioncomprises:

-   -   (i) at least one combination of amino acid modifications        selected from the group consisting of:    -   226G/330V, 230L/264E, 230L/378V, 230S/315D, 230S/434Y,        230T/378V, 241L/434S, 250A/434Y, 264E/378T, 305A/315D,        305A/330V, 305A/434Y, 307P/434Y, 315D/389T, 330V/382V,        330V/389T, 378V/421T, 389K/434Y, 389T/434Y, 396L/434S,        230T/264E, 230T/315D, 230T/434S, 230T/434Y, 241L/307P,        264E/307P, 264E/396L, 315D/362R, 315D/382V, 362R/434Y,        378V/434Y, 382V/434Y, 226G/315D, 226G/434Y, 241L/378V,        307P/378V, 241L/264E, 378V/434S, 264E/378V, 264E/434S,        315D/330V, 330V/434Y, and 315D/434Y; and    -   (ii) at least one amino acid modifications selected from the        group consisting of 226G, 227L, 228L, 228R 230S, 230T, 230L,        231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E,        294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V,        330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T,        382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L,        397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S        and 439R

of the Fc region, as compared to the parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modifications (i) does not occurat the same amino acid position as the modification (ii).

In other embodiments, the said variant comprises at least onecombination of amino acid modifications selected from the groupconsisting of 250A/434Y, 307P/434Y, 230T/434S, 264E/396L, 378V/434Y,378V/434S, 264E/378V, 264E/434S, 315D/330V, and 315D/434Y of the Fcregion, wherein the numbering of the amino acids in the Fc region isthat of the EU index as in Kabat.

The said variant may further comprise at least one amino acidmodification selected from the group of 226G, 226Y, 227S, 227L, 228R,228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L,241Y, 241R, 243L, 246R, 250A, 252L, 256N, 259I, 264A, 264E, 264M, 265G,265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y, 288R, 2891,290R, 290E, 291S, 291Q, 292W, 294del, 297D, 298G, 298N, 299M, 299A,299K, 301C, 302A, 303A, 3031, 305A, 307P, 307A, 307N, 308I, 309P, 311R,315D, 317R, 320T, 320E, 322R, 325S, 327V, 327T, 330V, 330T, 332V, 334E,334R, 335A, 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A,352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R, 361D, 361S, 362R, 362E,369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R,383N, 384I, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S,392E, 392R, 393N, 394A, 395A, 395S, 396S, 396L, 397A, 397M, 398P, 399N,400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A, 414R, 415D, 415N, 416K,416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422A, 424L, 426T,428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F,444P, 445S, 446A, 447E and 447N of the Fc region, as compared to theparent polypeptide, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat.

In another embodiment, a variant according to the present inventioncomprises:

-   -   (i) at least one combination of amino acid modifications        selected from the group consisting of:    -   250A/434Y, 307P/434Y, 230T/434S, 264E/396L, 378V/434Y,        378V/434S, 264E/378V, 264E/434S, 315D/330V, and 315D/434Y; and    -   (ii) at least one amino acid modifications selected from the        group consisting of 226G, 227L, 228L, 228R, 230S, 230T, 230L,        231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E,        294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V,        330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T,        382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L,        397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S        and 439R

of the Fc region, as compared to the parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modifications (i) does not occurat the same amino acid position as the modification (ii).

In some embodiments, the said variant comprises at least one amino acidcombination of modifications selected from the group consisting of:

226G/315D/330V, 226G/315D/434Y, 226G/330V/434Y, 230L/264E/378V,230T/264E/378V, 230T/264E/434S, 230S/315D/434Y, 230T/315D/434Y,230T/389T/434S, 241L/264E/434S, 241L/264E/378V, 241L/264E/307P,241L/307P/378V, 250A/389K/434Y, 256N/378V/434Y, 259I/315D/434Y264E/378T/396L, 264E/378V/416K, 294del/307P/434Y, 264E/307P/378V,264E/396L/434S, 264E/378V/434S, 305N315D/330V, 305A/315D/434Y,305A/330V/434Y, 307P/378V/434Y, 315D/330V/382V, 315D/330V/389T,315D/378V/434Y, 315D/389T/434Y, 315D/362R/434Y, 315D/382V/434Y,315D/330V/434Y 330V/382V/434Y, 330V/389T/434Y, and 378V/383N/434Y of theFc region, wherein the numbering of the amino acids in the Fc region isthat of the EU index as in Kabat.

The said variant may comprise at least one additional modificationselected from the group consisting of 226G, 226Y, 227S, 227L, 228R,228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L,241Y, 241R, 243L, 246R, 250A, 252L, 256N, 259I, 264A, 264E, 264M, 265G,265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y, 288R, 2891,290R, 290E, 291S, 291Q, 292W, 294del, 297D, 298G, 298N, 299M, 299A,299K, 301C, 302A, 303A, 3031, 305A, 307P, 307A, 307N, 308I, 309P, 311R,315D, 317R, 320T, 320E, 322R, 325S, 327V, 327T, 330V, 330T, 332V, 334E,334R, 335A, 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A,352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R, 361D, 361S, 362R, 362E,369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R,383N, 384I, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S,392E, 392R, 393N, 394A, 395A, 395S, 396S, 396L, 397A, 397M, 398P, 399N,400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A, 414R, 415D, 415N, 416K,416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422A, 424L, 426T,428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F,444P, 445S, 446A, 447E and 447N of the Fc region, as compared to theparent polypeptide, wherein the numbering of the amino acids in the Fcregion is that of the EU index as in Kabat.

In another embodiment, a variant according to the present inventioncomprises:

-   -   (i) at least one combination of amino acid modifications        selected from the group consisting of:    -   226G/315D/330V, 226G/315D/434Y, 226G/330V/434Y, 230L/264E/378V,        230T/264E/378V, 230T/264E/434S, 230S/315D/434Y, 230T/315D/434Y,        230T/389T/434S, 241L/264E/434S, 241L/264E/378V, 241L/264E/307P,        241L/307P/378V, 250A/389K/434Y, 256N/378V/434Y, 259I/315D/434Y        264E/378T/396L, 264E/378V/416K, 294del/307P/434Y,        264E/307P/378V, 264E/396L/434S, 264E/378V/434S, 305A/315D/330V,        305N315D/434Y, 305A/330V/434Y, 307P/378V/434Y, 315D/330V/382V,        315D/330V/389T, 315D/389T/434Y, 315D/362R/434Y, 315D/378V/434Y,        315D/382V/434Y, 315D/330V/434Y 330V/382V/434Y, 330V/389T/434Y,        and 378V/383N/434Y; and    -   (ii) at least one amino acid modification selected from the        group consisting of 226G, 227L, 228L, 228R, 230S, 230T, 230L,        231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E,        294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V,        330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T,        382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L,        397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S        and 439R

of the Fc region, as compared to the parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modifications (i) does not occurat the same amino acid position as the modification (ii).

In other embodiments, the said variant comprises at least one amino acidcombination of modifications selected from the group consisting of:

-   -   226G/315D/434Y, 230S/315D/434Y, 230T/315D/434Y, 230T/264E/434S,        230T/389T/434S, 241L/264E/378V, 241L/264E/434S, 250A/389K/434Y,        256N/378V/434Y, 259I/315D/434Y, 264E/378T/396L, 264E/378V/416K,        264E/378V/434S, 264E/396L/434S, 294del/307P/434Y,        307P/378V/434Y, 315D/330V/434Y, 315D/378V/434Y, 315D/382V/434Y        and 378V/383N/434Y of the Fc region, wherein the numbering of        the amino acids in the Fc region is that of the EU index as in        Kabat.

The said variant may further comprise at least one additionalmodification selected from the group consisting of 226G, 226Y, 227S,227L, 228R, 228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R,239A, 241L, 241Y, 241R, 243L, 246R, 250A, 252L, 256N, 259I, 264A, 264E,264M, 265G, 265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y,288R, 2891, 290R, 290E, 291S, 291Q, 292W, 294del, 297D, 298G, 298N,299M, 299A, 299K, 301C, 302A, 303A, 3031, 305A, 307P, 307A, 307N, 308I,309P, 311R, 315D, 317R, 320T, 320E, 322R, 325S, 327V, 327T, 330V, 330T,332V, 334E, 334R, 335A, 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G,347R, 350A, 352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R, 361D, 361S,362R, 362E, 369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V,382G, 383R, 383N, 384I, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K,389R, 390S, 392E, 392R, 393N, 394A, 395A, 395S, 396S, 396L, 397A, 397M,398P, 399N, 400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A, 414R, 415D,415N, 416K, 416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422A,424L, 426T, 428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N,443R, 444F, 444P, 445S, 446A, 447E and 447N of the Fc region, ascompared to the parent polypeptide, wherein the numbering of the aminoacids in the Fc region is that of the EU index as in Kabat.

In another embodiment, a variant according to the present inventioncomprises:

-   -   (i) at least one combination of amino acid modifications        selected from the group consisting of:    -   226G/315D/434Y, 230S/315D/434Y, 230T/315D/434Y, 230T/264E/434S,        230T/389T/434S, 241L/264E/378V, 241L/264E/434S, 250A/389K/434Y,        256N/378V/434Y, 259I/315D/434Y, 264E/378T/396L, 264E/378V/416K,        264E/378V/434S, 264E/396L/434S, 294del/307P/434Y,        307P/378V/434Y, 315D/330V/434Y, 315D/378V/434Y, 315D/382V/434Y        and 378V/383N/434Y; and    -   (ii) at least one amino acid modification selected from the        group consisting of 226G, 227L, 228R, 228L, 230S, 230T, 230L,        231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E,        294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V,        330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T,        382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L,        397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S        and 439R

of the Fc region, as compared to the parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index asin Kabat and with the proviso that the modifications (i) does not occurat the same amino acid position as the modification (ii).

In all previously cited embodiments, the said variant preferably hasfrom 1 to 20, more preferably from 1 to 10 amino acid modifications ascompared to the parent polypeptide.

In an alternate embodiment, the said variant comprises one combinationof amino acid modifications selected from the group consisting of307A/315D/330V/382V/389T/434Y, 307A/315D/382V/389T/434Y,256N/378V/383N/434Y, 256N/378V/434Y, 315D/330V/361D/378V/434Y,315D/361D/378V/434Y, 259I/315D/434Y, 230S/315D/428L/434Y,241L/264E/307P/378V/433R, 250A/389K/434Y, 305A/315D/330V/395A/434Y,264E/386R/396L/434S/439R, 315D/330V/362R/434Y, 294del/307P/434Y,305N315D/330V/389K/434Y, 315D/327V/330V/397M/434Y,230T/241L/264E/265G/378V/421T, 264E/396L/415N/434S, 227L/264E/378V/434S,264E/378T/396L, 230T/315D/362R/426T/434Y, 226G/315D/330V/434Y,230L/241L/243L/264E/307P/378V, 250A/315D/325S/330V/434Y,290E/315D/342R/382V/434Y, 241L/315D/330V/392R/434Y,241L/264E/307P/378V/434S, 230T/264E/403T/434S, 264E/378V/416K,230T/315D/362E/434Y, 226G/315D/434Y, 226G/315D/362R/434Y,226G/264E/347R/370R/378V/434S, 308I/315D/330V/382V/434Y,230T/264E/378V/434S, 231T/241L/264E/378T/397M/434S, 230L/264E/378V/434S,230T/315D/330V/386K/434Y, 226G/315D/330V/389T/434Y,267R/307P/378V/421T/434Y, 230S/315D/387T/434Y, 230S/264E/352S/378V/434Sand 230T/303A/322R/389T/404L/434S of Fc region, wherein the numbering ofthe amino acids in the Fc region is that of the EU index as in Kabat.

In other embodiment, the said variant comprises one combination of aminoacid modifications selected from the group consisting of 256N/378V/434Y,307A/315D/330V/382V/389T/434Y, 256N/378V/383N/434Y,315D/330V/361D/378V/434Y, 259I/315D/434Y and 230S/315D/428L/434Y.

A further object of the invention is to provide polypeptide variantswith increased binding for FcRn as compared to their parent polypeptidesand comprising a Fc variant selected from the group consisting of307A/315D/330V/382V/389T/434Y, 307A/315D/382V/389T/434Y256N/378V/383N/434Y, 315D/330V/361D/378V/434Y, 315D/361D/378V/434Y259I/315D/434Y, 230S/315D/428L/434Y, 241L/264E/307P/378V/433R,250N389K/434Y, 256N/378V/434Y, 305A/315D/330V/395A/434Y,264E/386R/396L/434S/439R, 315D/330V/362R/434Y, 294del/307P/434Y,305N315D/330V/389K/434Y, 315D/327V/330V/397M/434Y,230T/241L/264E/265G/378V/421T, 264E/396L/415N/434S, 227L/264E/378V/434S,264E/378T/396L, 230T/315D/362R/426T/434Y, 226G/315D/330V/434Y,230L/241L/243L/264E/307P/378V, 250A/315D/325S/330V/434Y,290E/315D/342R/382V/434Y, 241L/315D/330V/392R/434Y,241L/264E/307P/378V/434S, 230T/264E/403T/434S, 264E/378V/416K,230T/315D/362E/434Y, 226G/315D/434Y, 226G/315D/362R/434Y,226G/264E/347R/370R/378V/434S, 308I/315D/330V/382V/434Y,230T/264E/378V/434S, 231T/241L/264E/378T/397M/434S, 230L/264E/378V/434S,230T/315D/330V/386K/434Y, 226G/315D/330V/389T/434Y,267R/307P/378V/421T/434Y, 230S/315D/387T/434Y, 230S/264E/352S/378V/434Sand 230T/303A/322R/389T/404L/434S, wherein the numbering of the aminoacids in the Fc region is that of the EU index as in Kabat. In someother embodiments, the polypeptide variant with increased binding forFcRn as compared to its parent polypeptide comprises a Fc variantselected from the group consisting of 256N/378V/434Y,307A/315D/330V/382V/389T/434Y, 256N/378V/383N/434Y,315D/330V/361D/378V/434Y, 259I/315D/434Y and 230S/315D/428L/434Y.

For all the above-mentioned variants according to the invention, the Fcregion of their parent polypeptides may derive from the Fc regions ofwild-type IgGs (e.g “lower hinge-CH2-CH3”) and fragments thereof. In amore preferred embodiment, the Fc region of parent polypeptides derivesfrom the human IgG subclasses namely IgG1, IgG2, IgG3 and IgG4. Inanother preferred embodiment, the Fc region of the parent polypeptidesis selected from the group consisting of the wild-type IgG1 Fc region(SEQ ID NO:1), the wild-type IgG2 Fc region (SEQ ID NO:2), the wild-typeIgG3 Fc region (SEQ ID NO:3) and the wild-type IgG4 Fc region (SEQ IDNO:4).

In this context, another object of the invention is a polypeptidecomprising a IgG1 Fc variant wherein said IgG1 Fc variant comprises atleast one amino acid modification as compared to the wild-type sequenceof IgG1 Fc (SEQ ID NO:1) and displays an increased binding to FcRn ascompared to the wild-type IgG1 Fc with the proviso that the sequence ofsaid IgG1 Fc variant is not SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

Another object of the invention is a polypeptide comprising an IgG2 Fcvariant wherein said IgG2 Fc variant comprises at least one amino acidmodification as compared to the wild-type sequence of IgG2 Fc (SEQ IDNO:2) and displays an increased binding to FcRn as compared to thewild-type IgG2 Fc with the proviso that the sequence of said IgG2 Fcvariant is not SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4.

An additional object of the invention is a polypeptide comprising anIgG3 Fc variant wherein said IgG3 Fc variant comprises at least oneamino acid modification as compared to the wild-type sequence of IgG3 Fc(SEQ ID NO:3) and displays an increased binding to FcRn as compared tothe wild-type IgG3 Fc with the proviso that the sequence of said IgG3 Fcvariant is not SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4.

Another object of the invention is a polypeptide comprising an IgG4 Fcvariant wherein said IgG4 Fc variant comprises at least one amino acidmodification as compared to the wild-type sequence of IgG4 Fc (SEQ IDNO:4) and displays an increased binding to FcRn as compared to thewild-type IgG4 Fc with the proviso that the sequence of said IgG2 Fcvariant is not SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:2.

In preferred embodiments, the at least one amino acid modificationcomprised in the IgG1, IgG2, IgG3 or IgG4 Fc variant polypeptides areselected from the group of amino acid modifications and combinations ofamino acid modifications that are described above in the instantspecification when generally defining the variant of a polypeptidecomprising an Fc region and having an increased binding to FcRn ascompared to the corresponding parent polypeptide.

As described above, a variant according to the invention exhibits anincreased binding to FcRn as compared to the corresponding parentpolypeptide. In one embodiment, the effector functions and the otherbinding properties of the said variant are similar to that of thecorresponding parent. The said variant may particularly exhibit nosignificant change in binding to Fc-gamma receptors or C1q as comparedto its parent polypeptide.

In another embodiment, the said variant has an increased binding to FcRncombined with one or more altered effector functions and/or binding toFc ligands (other than FcRn).

As illustrated in Example 2, the variant of the invention may have anincreased binding to FcRn combined with unaltered binding to a FcγR, inparticular to FcγRIIIa, ADCC (Antibody-Dependent Cell-mediatedCytotoxicity) activity and CDC (Complement-Dependent Cytotoxicity)activity as compared to the polypeptide variant. The variant of theinvention may also have an increased binding to FcRn combined with ADCCand CDC activities which are at least similar to that of its polypeptideparent. In some other cases, the variant of the invention may have anincreased binding to FcRn combined with at least one reduced effectoractivity selected from ADCC and CDC as compared to its polypeptideparent.

ADCC and CDC activities may be assessed by well-known methods of theprior art such as those described in Example 2 parts IV.2 and IV.3 ofthe present specification.

The binding to FcγR may be assessed by conventional methods such as SPRor ELISA assay.

A further object of the invention is to provide variants whichoptionally comprise additional amino acid modifications which differfrom those cited previously with the proviso that the resulting variantshave an increased binding to FcRn as compared to the parent polypeptide.

Accordingly, the Fc modifications of the present invention may becombined with other Fc modifications which are known to increase the Fcaffinity for FcRn (see for example the references cited in the part ofthe present application dedicated to the description of the relatedart).

Alternatively, the Fc modifications may be combined with other Fcmodifications including but not limited to modifications that altereffector function or interaction with one or more Fc ligands. As aconsequence, such variants may display an increased binding to FcRncombined with an altered binding to one Fc ligand (other than FcRn)or/and an altered effector function as compared to the parentpolypeptide.

Fc ligands include but are not limited to FcγRs (Fcgamma. receptors),C1q, C3, mannan binding lectin, mannose receptor, staphylococcal proteinA, streptococcal protein G, and viral FcγRs. Fc ligands also include Fcreceptor homologs (FcRH), which are a family of Fc receptors that arehomologous to the FcgammaRs (Davis et al., 2002, Immunological Reviews190:123-136).

By “effector function” as used herein is meant a biochemical or cellularevent that results from the interaction of an antibody Fc region with anFc receptor or ligand. Effector functions include but are not limited toADCC (Antibody-Dependent Cell-mediated Cytotoxicity), ADCP(Antibody-Dependent Cell-mediated Phagocytosis), and CDC (ComplementDependent Cytotoxicity).

The variants of the present invention encompass any polypeptidecomprising an Fc region and displaying an increased binding affinity forFcRn as compared to its parent polypeptide with the proviso that thesaid polypeptide differs from its parent polypeptide in virtue of atleast one amino acid modification or combination of amino acidmodifications in the Fc region. The modifications and the combinationsof amino acid modifications of interest are those described above whengiving general features of the variants according to the invention.

The variants (and thus the parent polypeptides) include, but are notlimited to, antibodies, Fc fusion proteins, Fc conjugates, isolated Fcand their fragments respectively. In particular, the variants can be anFc-comprising binding protein. In other words, the variants comprising(i) an Fc variant and (ii) a binding polypeptide domain which is able tospecifically bind to a given molecule.

In one embodiment, the polypeptide variants of the invention areselected from the group consisting of Fc-fusion protein variants andFc-conjugate variants. Fc-fusion protein and Fc-conjugates consist of anFc region linked to a partner. The Fc region can be linked to itspartner with or without a spacer.

According to the present invention, an Fc fusion protein is a proteinencoded by a single gene and comprises a protein, a polypeptide or asmall peptide linked to an Fc region. An Fc fusion protein optionallycomprises a peptide spacer. Virtually any protein or small molecule maybe linked to Fc regions to generate an Fc fusion. Protein fusionpartners may include, but are not limited to, the variable region of anyantibody, a polypeptide derived from a variable region of any antibody,the target-binding region of a receptor, an adhesion molecule, a ligand,an enzyme, a cytokine, a chemokine, or some other protein or proteindomain. In particular the Fc-fusion protein can be an immunoadhesin i.eantibody-like protein which combines the binding domain of aheterologous “adhesion” protein (i.e receptor, ligand or enzyme) with afragment of immunoglobulin constant domain (i.e. an Fc region) (see fora review about immunoadhesins, Ashkenazi A, Chamow S M. 1997, Curr OpinImmunol.; 9 (2):195-200).

Small peptide fusion partners may include, but are not limited to, anytherapeutic agent that directs the Fc fusion to a therapeutic target.Such targets may be any molecule, preferably an extracellular receptorthat is implicated in disease.

According to the present invention, an Fc conjugate results from thechemical coupling of a Fc region with a conjugate partner. The conjugatepartner can be proteinaceous or non-proteinaceous. The coupling reactiongenerally uses functional groups on the Fc region and on the conjugatepartner. Various linkers are known in the art to be appropriate for thesynthesis of conjugate; for example, homo- or hetero-bifunctionallinkers are well known (see, Pierce Chemical Company catalog, 2005-2006,technical section on cross-linkers, pages 321-350, incorporated hereinby reference.

Suitable conjugate partners include, but are not limited to, therapeuticpolypeptides, labels (for example of labels, see further below), drugs,cytotoxic agents, cytotoxic drugs (e.g., chemotherapeutic agents),toxins and active fragments of such toxins. Suitable toxins and theircorresponding fragments include, but are not limited to, diptheria Achain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin,phenomycin, enomycin and the like. A cytotoxic agent may be anyradionuclide which can be directly conjugated to the Fc variant orsequestrated by a chelating agent which is covalently attached to the Fcvariant. In additional embodiments, the conjugate partners can beselected from the group consisting of calicheamicin, auristatins,geldanamycin, maytansine, and duocarmycins and analogs; (for the latter,see U.S. 200310050331, hereby incorporated by reference in itsentirety).

Such variants of interest may have an increased binding to FcRn atlowered pH (e.g at about pH 6), and substantially unmodified binding athigher pH (e.g. at about pH 7.4). Of particular interest are Fc-fusionprotein and Fc-conjugate variants which display increased in vivohalf-lives as compared to parent polypeptides.

In a preferred embodiment, the polypeptide variant of the presentinvention is a variant antibody of a parent antibody. The term“antibody” is used herein in the broadest sense. According to thepresent invention, “antibody” refers to any polypeptide which at leastcomprises (i) a Fc region and (ii) a binding polypeptide domain derivedfrom a variable domain of an immunoglobulin. The said bindingpolypeptide domain is able to bind specifically one given target antigenor a group of target antigens. A binding polypeptide domain whichderives from a variable region of an immunoglobulin comprises at leastone or more CDRs. Herein, antibodies include, but are not limited to,full-length immunoglobulins, monoclonal antibodies, multi-specificantibodies, Fc-fusion protein comprising at least one variable region,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, humanized antibodies and fully humanantibodies. Antibodies also encompass antibody-fusion proteins, antibodyconjugates and fragments of each respectively. Accordingly a variantantibody of the invention comprises, in its Fc region, at least oneamino acid modification or combination of modifications above-cited thatincrease its binding affinity for FcRn as compared to its parentantibody. Of particular interest are antibody variants that displayincreased binding affinity to FcRn at lowered pH (e.g at about pH 6),and have substantially unmodified binding at higher pH (e.g. at about pH7.4). Furthermore, of particular interest are antibody variants whichhave increased in vivo half-lives as compared to parent polypeptides.

In one embodiment, a variant antibody of the invention is selected fromthe group consisting of variants of parent full-length antibodies. By“full length antibody” herein is meant the structure that constitutesthe natural biological form of an antibody, including variable andconstant regions. The parent polypeptide of a full-length antibodyvariant of the present invention can be a wild-type antibody, a mutantof a wild-type antibody (e.g. comprising pre-existing modifications), anengineered version of a wild-type antibody (e.g. for example a chimeric,a humanized antibody or a fully human antibody, see further below), thislist not being limitative. The structure of a full-length antibody isgenerally a tetramer except for some mammals such as llamas and camelsin which some immunoglobulins are dimers. Each tetramer is typicallycomposed of two identical pairs of polypeptide chains, each pair havingone “light” (typically having a molecular weight of about 25 kDa) andone “heavy” chain (typically having a molecular weight of about 50-70kDa).

Examples of full-length antibodies are human immunoglobulins whichencompass IgM, IgD, IgG, IgA and IgE classes.

In preferred embodiments, the said full-length antibody variant isselected from the group consisting of variants of IgGs.

In more preferred embodiments, the said full-length antibody variant isselected from the group consisting of variants of human IgG1, IgG2, IgG3and IgG4 with the proviso that the said Fc region sequence of saidvariant is not SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

The said IgG variant comprises one or more amino acid modifications ascompared to its parent IgG, said one or more modifications orcombinations of amino acid modifications are those previously describedin the present specification when generally defining the variants of apolypeptide comprising a Fc region and having an increased binding toFcRn as compared to the corresponding parent polypeptide.

In another embodiment, the said antibody variant is selected from thegroup consisting of Fc-fusion protein comprising a binding polypeptidedomain derived from a variable domain of an immunoglobulin. Ofparticular interest are antibodies that comprise (a) a Fc variant of theinventions, and (b) one of the following binding polypeptide domainsderived from a variable region of an immunoglobulin (i.e. which compriseat least one CDR): (i) the Fab fragment consisting of VL, VH, CL and CH1domains, (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) isolated CDR regions, (v) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vi) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site, (vii) bispecific single chain Fv and (viii)“diabodies” or “triabodies”, multivalent or multispecific fragmentsconstructed by gene fusion, this list not being limitative.

In another embodiment, the antibody is a minibody. Minibodies areminimized antibody-like proteins comprising a scFv joined to a CH3domain (Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated byreference herein in its entirety). In some cases, the scFv can be joinedto a full-length Fc region (De Lorenzo et al., 2005, Carcinogenesis26:1890-1895, incorporated by reference herein its entirety), and mayalso include the hinge region or fragment thereof.

In one embodiment, the antibodies of the invention are selected from thegroup of multispecific antibodies, and notably from the group ofbispecific antibodies which are sometimes referred to as “diabodies”.These antibodies bind to two (or more) different antigens. Diabodies canbe manufactured in a variety of ways known in the art (Holliger andWinter, 1993, Current Opinion Biotechnol. 4:446-449, incorporated byreference herein in its entirety), e.g., chemically prepared or derivedfrom hybridomas.

In some embodiments, the scaffold components of the antibody variantscan be a mixture from different species. Such antibody variant may be achimeric antibody and/or a humanized antibody. In general, both“chimeric antibodies” and “humanized antibodies” refer to antibodiesthat combine regions from more than one species. For example, “chimericantibodies” traditionally comprise variable region(s) from a non-humananimal, generally the mouse (or rat, in some cases) and the constantregion(s) from a human. For the most part, humanized antibodies arechimeric antibodies that contain minimal sequence derived from non humanimmunoglobulin. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to a human antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018; Jones, 1986, Nature 321:522-525;Verhoeyen et al., 1988, Science 239: 1534-1536, all incorporated byreference herein in their entirety. The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system (Roqueet al., 2004, Biotechnol. Prog. 20:639-654, incorporated by referenceherein in its entirety). A variety of techniques and methods forhumanizing and reshaping non-human antibodies are well known in the art(See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies,Molecular Biology of B Cells, 533-545, Elsevier Science (USA), andreferences cited therein, all incorporated by reference in theirentirety). Humanization methods include but are not limited to methodsdescribed in Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9; Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, allincorporated by reference in their entirety. Humanization or othermethods of reducing the immunogenicity of nonhuman antibody variableregions may include resurfacing methods, as described for example inRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973,incorporated by reference herein in its entirety.

In one embodiment, the said antibody variant is a fully human antibodywith at least one amino acid modification as outlined herein. “Fullyhuman antibody” or “complete human antibody” refers to an antibodyentirely comprising sequences originating from human genes. In somecases this may be human antibodies that have the gene sequence of anantibody derived from a human chromosome with the modifications outlinedherein. Alternatively, the components of the antibody may be human butnot be derived from a single gene. Thus, for example, human CDRs fromone antibody can be combined with sequences, such as scaffold sequences,from one or more human antibodies. For example, a variety of germlinesequences can be combined to form a human antibody or human scaffold(e.g. for use in humanized or chimeric sequences as outlined above), aswell as U.S. patent application Ser. No. 11/022,289, incorporated hereinby reference in its entirety.

In certain embodiments, the antibody variant of the invention isselected from the group consisting of chimeric IgGs, humanized IgGs andfully-human IgGs.

Covalent modifications of antibodies are also included within the scopeof this invention, and are generally, but not always, donepost-translationally. Such modifications include, but are not limitedto, glycosylations, labelling and conjugation.

Accordingly, in some embodiments, the polypeptide variants disclosedherein can be modified to include one or more engineered glycoforms. By“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to the polypeptide comprisingthe Fc variant, wherein said carbohydrate composition differs chemicallyfrom that of a polypeptide parent. Engineered glycoforms may be usefulfor a variety of purposes, including but not limited to enhancing orreducing effector function. The engineered glycoforms can be attached atany amino acid of the variant sequence. In a preferred embodiment, thesaid glycoforms are attached at amino acids of the Fc region.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. Nos. 10/277,370; 10/113,929; WO00/61739A1; WO 01/29246A1; WO 02/31140A1; WO 02/30954A1, WO 01/77181,all incorporated by reference in their entirety; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zuerich,Switzerland]). Many of these techniques are based on controlling thelevel of fucosylated and/or bisecting oligosaccharides that arecovalently attached to the Fc region, for example by expressing theantibody variant in various organisms or cell lines, engineered orotherwise (for example Lec-13 CHO cells or rat hybridoma YB2/0 cells),by regulating enzymes involved in the glycosylation pathway (for exampleFUT8 [α1,6-fucosyltranserase] and/or(β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the antibody variant has been expressed.

Alternatively, engineered glycoform may refer to the antibody variantthat comprises the different carbohydrate or oligosaccharide. As isknown in the art, glycosylation patterns can depend on both the sequenceof the protein (e.g., the presence or absence of particularglycosylation amino acid residues, discussed below), or the host cell ororganism in which the protein is produced. Particular expression systemsare discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

In some embodiments, the antibody variant of the invention is selectedfrom the group consisting of chimeric IgGs, humanized IgGs andfully-human IgGs which comprise engineered glycoforms.

In an alternative embodiment, the covalent modification of the antibodyvariants of the invention comprises the addition of one or more labels.In some cases, these are considered antibody fusions. The term “labelinggroup” means any detectable label. In some embodiments, the labelinggroup is coupled to the antibody via spacer arms of various lengths toreduce potential steric hindrance. Various methods for labeling proteinsare known in the art and may be used in performing the presentinvention.

In general, labels fall into a variety of classes, depending on theassay or on the diagnostic procedure in which they are to be detected:a) isotopic labels, which may be radioactive or heavy isotopes; b)magnetic labels (e.g., magnetic particles); c) redox active moieties; d)optical dyes; enzymatic groups (e.g. horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase); e) biotinylatedgroups; and f) predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags, etc).

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either fluorescent “smallmolecules” fluorescent, or fluorescent proteins.

In another embodiment, the antibody variants of the present inventionmay be fused to or conjugated to a protein or a small molecule which arenot used as a labelling group as described above. Virtually any proteinor small molecule may be linked to an antibody. Protein fusion partnersmay include, but are not limited to, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculesinclude, but are not limited to drugs, cytotoxic agents (e.g.,chemotherapeutic agents), toxins or active fragments of such toxins.

As described above, the antibody variants of the invention are able tobind specifically one target antigen or a group of target antigens. By“target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody orimmunoglobulin. A target antigen may be a protein, a carbohydrate, alipid, or other chemical compound.

The choice of suitable antigen depends on the desired application.Virtually, any antigen may be targeted, for example membrane proteinscomprising but not limited to the RhD antigen, CD3, CD4, CD19, CD20,CD22, CD25, CD28, CD32B, CD33, CD38, CD40, CD44, CD52, CD71 (transferrinreceptor), CD80, CD86, CTLA-4, CD147, CD160, CD224, growth factorreceptors like those belonging to the ErbB family of receptors ErbB1,ErbB2, ErbB3, ErbB4 (EGFR, HER2/neu, HER3, HER4), VEGF-R1, VEGF-R2,IGF-R1, PIGF-R, MHC class I and MHC class II molecules, e.g. HLA-DR,interleukin receptors like IL-1R, IL-2R alpha, IL-2R beta and IL-2Rgamma, IL-6R, hormone receptors like Müllerian inhibitory substance typeII receptor, LDL receptor, NKp44L, chemokine receptors like CXCR4 andCCR5, integrins, adhesion molecules like CD2, ICAM, EpCAM. The membraneproteins also include tumour markers like GD2, GD3, CA125, MUC-1,MUC-16, carcinoembryonic antigen (CEA), Tn, glycoprotein 72, PSMA,HMW-MAA. Antibodies of the invention can also target soluble proteins,including but not limited to cytokines (for instance IL-1 beta, IL-2,IL-6, IL-12, IL-23, TGF beta, TNF alpha, IFN gamma), chemokines, growthfactors like VEGF-A, EGF, PIGF, PDGF, IGF, hormones, bacterial toxinsand toxins of other origin like botulinus toxin, ricin, B. anthracisprotective antigen, B. anthracis lethal factor, B. anthracis edemafactor, shigatoxins 1 and 2, viral antigens from different viruses, forexample pathogenic viruses, an inhibitory antibody, including a FVIIIinhibitory antibody.

In a preferred embodiment, the variant of the present invention maytarget CD20. In this case, the parent polypeptide can be selected from:EMAB6 or EMAB603 (see WO2006064121), RITUXIMAB (Rituxan®,IDEC/Genentech/Roche) (see for example U.S. Pat. No. 5,736,137,incorporated by reference in its entirety), HUMAX®-CD20, described inU.S. Pat. No. 5,500,362 incorporated by reference in its entirety,AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.),HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”, incorporated by reference inits entirety).

In another embodiment, the variant of the present invention may targetRhD antigen. In this case, the parent polypeptide can be selected fromEMAB2 (see FR 03 12 229), Sym001 (Symphogen A/S) or MonoRho (ZLB,Zurich).

The parent polypeptide may also be Avastin® (anti-VEGF), Remicade®(anti-TNF-α), Erbitux®, Vectibix® (anti-EGFR), Tysabri® (anti-alpha4chain of integrine), Herceptin® (anti-HER2/neu), the list not beinglimitative.

The present application also provides variants that display an increasedbinding to FcRn combined with another optimized property selected from avariety of well-known therapeutically relevant properties. The mostpreferred property that may be optimized is the in vivo half-life. Todisplay an increased in vivo half-life, the variant should exhibitincreased binding affinity to FcRn at lower pH, such as the pHassociated with endosomes, e.g. pH 6.0, while maintaining the reducedaffinity at higher pH, such as 7.4, to allow increased binding to FcRninto endosomes but normal release rates (Dall'Acqua et al., 2002, J.Immunol. 169: 5171-5180; Gurbaxani et al., 2006, Mol Immunol. 43(9):1462-73). Similarly, these variants with such modulated FcRn bindingmay optionally have other desirable properties, such as modulated FcγRbinding. In one additional embodiment, the variants are optimized topossess enhanced affinity for a human activating FcγR, preferablyFcγRIIIa in addition to the FcRn binding profile. In an alternateembodiment, the variants are optimized to have increased affinity forFcRn and increased or decreased affinity for a human FcγR, including butnot limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and FcγRIIIb includingtheir allelic variations. In alternative embodiments, the variants ofthe present invention may optionally have increased (or decreased)effector functions as well as an increased serum half-life. Inparticularly preferred embodiments, a variant of the invention may haveincreased ADCC activity and/or increased binding to a FcγR as well asincreased serum half-life as compared to its polypeptide parent. Inother embodiments, the variant of the invention may further have anincreased CDC activity as compared to its polypeptide parent.

The variants may find use in a wide range of products. In one embodimentthe variant is a therapeutic, a diagnostic, or a research reagent,preferably a therapeutic.

Since they display increased binding to FcRn, the variant of theinvention are anticipated to have longer in vivo half-lives, moreprecisely longer in vivo serum half-lives than their parentpolypeptides. As a consequence, such variants have useful applicationsas parent polypeptide substitutes when the parent polypeptide is toorapidly cleared from the blood circulation or for use in the treatmentof chronic or long-term diseases which requires long half-life activeprinciples.

When the variants are selected from the group of antibodies, they mayfind use in an antibody composition that is monoclonal or polyclonal. Ina preferred embodiment, the said antibody variants are used to killtarget cells that bear the target antigen, for example cancer cells. Inan alternate embodiment, the variants are used to block, antagonize oragonize the target antigen, for example for antagonizing a cytokine orcytokine receptor, for neutralizing an infectious agent like a bacteriumor a virus or a toxin, for example, a bacterial toxin. In an alternatelypreferred embodiment, the variants are used to block, antagonize oragonize the target antigen and kill the target cells that bear thetarget antigen.

In a preferred embodiment, a variant antibody is administered to apatient to treat an antibody-related disorder. A “patient” for thepurposes of the present invention includes humans and other animals,preferably mammals and most preferably humans. By “antibody relateddisorder” or “antibody responsive disorder” or “condition” or “disease”herein are meant a disorder that may be ameliorated by theadministration of a pharmaceutical composition comprising a variant ofthe present invention. Antibody related disorders include but are notlimited to autoimmune diseases, immunological diseases, infectiousdiseases, inflammatory diseases, neurological diseases, pain, pulmonarydiseases, hematological conditions, fibrotic conditions, and oncologicaland neoplastic diseases including cancer. By “cancer” and “cancerous”herein refer to or describe the physiological condition in mammals thatis typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to carcinoma, lymphoma, blastoma,sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma,schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia andlymphoid malignancies. Other conditions that may be treated include butare not limited to rheumatoid arthritis, juvenile rheumatoid arthritis,Crohn's disease, ulcerative colitis, Sjorgren's disease, multiplesclerosis, ankylosing spondylitis, asthma, allergies and allergenicconditions, graft versus host disease, and the like.

A further object of the invention is to provide pharmaceuticalcompositions comprising the said variant. The said formulations areprepared by mixing the polypeptide variant having the desired degree ofpurity with optional physiologically acceptable pharmaceuticallyacceptable carrier, excipients or stabilizers in the form of lyophilisedformulations or aqueous solutions. (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed., 1980, incorporated by reference herein inits entirety). Such pharmaceutical compositions are destined fortreating a patient in need.

In order to treat a patient in need, a therapeutically effective dose ofthe variant may be administered. By “therapeutically effective dose”herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.001 to 100 mg/kg of bodyweight or greater, for example 0.1, 1.0, 10, or 50 mg/kg of body weight,with 1 to 10 mg/kg being preferred. As is known in the art, adjustmentsfor protein degradation, systemic versus localized delivery, and rate ofnew protease synthesis, as well as the age, body weight, general health,sex, diet, time of administration, drug interaction and the severity ofthe condition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

Administration of the pharmaceutical composition comprising a variantmay be done in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, parenterally, intranasally,intraortically, intraocularly, rectally, vaginally, transdermally,topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary.

Therapeutic described herein may be administered with other therapeuticsconcomitantly, i.e., the therapeutics described herein may beco-administered with other therapies or therapeutics, including forexample, small molecules, other biologicals, radiation therapy, surgery,etc.

Another object of the present invention is to provide isolated nucleicacids encoding variants according to the invention. Most often, the DNAencoding the parent polypeptide is available or can be obtained.Consequently, the DNA encoding the variant of interest can be generatedby altering the DNA encoding parent polypeptide thanks to a variety ofmethods known in the prior art. These methods include, but are notlimited to site-directed mutagenesis, random mutagenesis, PCRmutagenesis and cassette mutagenesis. Amino acid substitutions arepreferably made by site-directed mutagenesis (see, for example, Zollerand Smith, 1982, Nucl. Acids Res. 10:6487-6500; Kunkel, 1985, Proc.Natl. Acad. Sci USA 82:488, which are hereby incorporated by referencein their entireties).

Alternatively or additionally, the desired amino acid sequence encodinga polypeptide variant can be determined and thus can be generatedsynthetically by well-known methods of the prior art.

Once their encoding nucleic acids are obtained, the variants of thepresent invention can be made by any method known in the art. In oneembodiment, the variant sequences (e.g. IgG variant sequences) are usedto create nucleic acids that encode the member sequences, and that maythen be cloned into host cells, expressed and assayed, if desired. Thesepractices are carried out using well-known procedures, and a variety ofmethods that may find use in are described in Molecular Cloning—ALaboratory Manual, 3^(rd) Ed. (Maniatis, Cold Spring Harbor LaboratoryPress, New York, 2001), and Current Protocols in Molecular Biology (JohnWiley & Sons), both incorporated by reference in their entirety. Thenucleic acids that encode the variants may be incorporated into anexpression vector in order to express the protein. Expression vectorstypically include a protein operably linked, that is, placed in afunctional relationship, with control or regulatory sequences,selectable markers, any fusion partners, and/or additional elements. Thevariant (e.g. IgG variants) of the present invention may be produced byculturing a host cell transformed with nucleic acid, preferably anexpression vector, containing nucleic acid encoding the variant, underthe appropriate conditions to induce or cause expression of the protein.A wide variety of appropriate host cell lines may be used, including butnot limited to mammalian cells, bacteria, insect cells, and yeast. Forexample, a variety of mammalian cell lines that may find use aredescribed in the ATCC cell line catalog, available from the AmericanType Culture Collection. Host cells may be, but not limited to, YB2/0(YB2/3HL.P2.GII.IGAg.20 cell, deposit to the American Type CultureCollection, ATCC no CRL-1662), SP2/0, YE2/0, 1R983F, Namalwa, PERC6, CHOcell lines, particularly CHO-K-1, CHO-Lecl0, CHO-Lecl, CHO-Lecl3, CHOPro-5, CHO dhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK,KGH6, NSO, SP2/0-Ag 14, P3X63Ag8.653, C127, JC, LA7, ZR-45-30, hTERT,NM2C5, UACC-812 and the like. The methods of introducing exogenousnucleic acid into host cells are well known in the art, and will varywith the host cell used. In a preferred embodiment of the invention, thevariant is expressed in YB2/0 cell, and is an anti-CD20 antibody, or ananti-RhD antibody.

In addition, a variant according to the present invention may beproduced by a transgenic non-human animal or transgenic plant. Also, atransgenic non-human animal can be obtained by directly injecting adesired gene into a fertilized egg (Gordon et al., 1980 Proc Natl AcadSci USA.; 77:7380-4). The transgenic non-human animals include mouse,rabbit, rat, goat, cow, cattle or fowl, and the like. A transgenicnon-human animal having a desired gene can be obtained by introducingthe desired gene into an embryonic stem cell and preparing the animal byan aggregation chimera method or injection chimera method (Manipulatingthe Mouse Embryo, A Laboratory Manual, Second edition, Cold SpringHarbor Laboratory Press (1994); Gene Targeting, A Practical Approach,IRL Press at Oxford University Press (1993)). Examples of the embryonicstem cell include embryonic stem cells of mouse (Evans and Kaufman,1981, Nature; 292:154-156), rat, goat, rabbit, monkey, fowl, cattle andthe like. In addition, a transgenic non-human animal can also beprepared using a clonal technique in which a nucleus into which adesired gene is introduced is transplanted into an enucleated egg (Ryanet al., 1997 Science; 278: 873-876; Cibelli et al., 1998 Science, 280:1256-1258). The polypeptide variant can be produced by introducing DNAencoding the variant molecule into an animal prepared by the abovemethod to thereby form and accumulate the variant molecule in theanimal, and then collecting the polypeptide variant from the animal. Thepolypeptide variant may be made to be formed and accumulated in themilk, egg or the like of the animal.

Another object of the invention is to provide a method for identifyingFc variants which are optimized variants i.e. which have an increasedbinding for an Fc ligand as compared to a corresponding wild-type Fcregion. Said method comprising the steps of:

-   -   (i) generating a nucleic acid library consisting of a set of        nucleic acids encoding Fc variants    -   (ii) producing the Fc variants by the expression of the nucleic        acids comprised in the said library    -   (iii) selecting among the Fc variants produced in step (ii),        those which are able to bind to the Fc ligand    -   (iv) measuring the binding property of the Fc variants selected        in step (iii) and that of one Fc control for the Fc ligand and    -   (v) selecting the Fc variants which display an increased binding        for the Fc ligand as compared to the said Fc control

The nucleic acid sequences comprised in the said nucleic acid librarymay be RNA or DNA. In a preferred embodiment, the said library comprisesDNA sequences encoding Fc variants.

The Fc control is selected from the group consisting of wild-type Fcregions and known Fc variants which have binding property for the Fcligand equal or higher than that of wild-type Fc.

The Fc ligand can be selected from FcRn and Fc.gamma.receptors, the listnot being limitative.

In one embodiment, the nucleic acids of the library which encode Fcvariants can be generated by altering the DNA sequence encoding for thewild-type Fc. As used herein, by “alter the nucleic acid sequence” ismeant the introduction of mutations such as insertions, deletions orsubstitutions of nucleotides in a given nucleic acid sequence. Suchmutations can be performed by well-known methods of the prior art. Thesemethods include, but are not limited to, random mutagenesis,site-directed mutagenesis, PCR mutagenesis and cassette mutagenesis.

In a preferred embodiment, the library is generated by randommutagenesis based on the use of one or more low fidelity DNApolymerases. Such random mutagenesis is described in the PCT applicationWO0238756 incorporated by reference in its entirety. Accordingly, thelibrary may be generated by the mixing of sub-libraries generated withone single polymerase or a specified combination of polymerases asdescribed in the material and methods part of the example of the presentapplication.

In another embodiment, the said nucleic acid library is generated byaltering the DNA sequences encoding for a pool of pre-optimized Fcvariants using one of the above-mentioned methods. Random mutagenesis ispreferably used. Pre-optimized Fc variants are Fc variants whichcomprise at least one amino acid modification and display an increasedbinding for the Fc ligand as compared to the wild-type Fc. Pre-optimizedFc variants have preferably 1 to 4 amino acid modifications as comparedto the wild-type Fc. Such pre-optimized Fc variants can be obtained fromthe screening of a library generated by mutation of a wild-type Fc. Theyalso refer to Fc variants described in the prior art (for examples seeabove the first part of the present application dedicated to thedescription of the related art). The pool of pre-optimized Fc variantscomprises several polypeptides, more preferably from about 2 to about100 pre-optimized variants.

The libraries generated from pre-optimized variants enable to selectmore optimized Fc variants. For illustration see table 5 of the presentapplication which shows the binding affinity of the best Fc variantsselected from the screening of such a library.

Step (ii) i.e the expression of Fc variants can be performed bywell-known methods using host cells as described previously. In apreferred embodiment the Fc library is expressed on the surface ofbacteriophages (phage display) using standard procedures (see Smith,Science, 1985, 228: 1315).

Step (iii) can be performed by generating Fc variants-Fc ligandcomplexes and then separating the bound Fc variants from the unbound Fcvariants. In order to perform this separation step, the Fc ligand may beadvantageously immobilized on a solid support or should be able to beimmobilized on a solid support during the process of step (iii).Examples of such procedures are described in Example 1 of the presentapplication. The step (iii) preferably comprises several rounds ofselection which enable to identify the most effective Fc ligands (forillustration see Example 2).

In step (iv), the binding properties of Fc variants for Fc ligand can beevaluated by well-known methods of the prior art. For example, the oneskilled in the art may perform an appropriate ELISA. The variant isselected if its specific signal is at least 1.2-fold stronger than thatof the Fc parent. Appropriate ELISA assays can be performed on isolatedFc or on Fc displayed on phage as illustrated in example II and inexample IV of the present application.

As an alternative or for confirmation purpose, the one skilled in theart may determine the dissociation constant using Surface PlasmonResonance (SPR) experiments as illustrated in the example IV of thepresent application. If the variant has a dissociation constant 3-foldlower then that of the Fc parent then the said variant is selected instep (v).

The present invention is further illustrated by, without on any waybeing limited to, the examples below.

EXAMPLES Example 1 Identification of Fc Variants with Increased Bindingto FcRn as Compared to Fc-Wild-Type and Binding Characterization of SaidVariants

I. Material and Methods

I.1. Expression and Purification of Human FcRn

The expression of soluble human FcRn using the baculovirus system wasperformed by GTP Technology (Labège, France) as previously described(Popov et al., Mol. Immunol. 33:521-530 (1996)). The α chain cDNAencoding the leader peptide and extracellular domains (codons 1-290) wastagged, with a TEV sequence and a 6× polyhistidine tag. The derivativeα-chain and the β2 microglobulin chain were cloned into pFastBacDualunder the P10 and polyhedrine promoters, respectively. A biotinylatedversion of FcRn (FcRn-biot) was prepared by chemical coupling with theFluoReporter® Biotin-XX Protein Labeling Kit, F2610 (Molecular Probes)according to the manufacturer's protocol. A fusion protein was alsoproduced containing the β2 microglobulin chain and the α-chain fused tothe amino terminal part of the bacteriophage β3 protein and the CVDEprotein (FcRn-p3). More than 90% pure proteins were obtained afterIgG-Sepharose and IMAC purification steps.

I.2. Construction of the Fc Libraries

Human Fc gene encoding amino acid residues 226-447 (EU index as inKabat) i.e. Fc polypeptide, derived from a human IgG1 heavy chain (SEQno 1), (Poul M A et al., Eur. J. Immunol. 25 (7): 2005-2009 (1995) wascloned into the phagemid vector pMG58 (pMG58_Fc226, FIG. 1) as aBamHI/EcoRI fragment using standard PCR protocols. The said vector isdepicted in FIG. 1. Several fully randomised libraries were generatedusing the MUTAGEN™ procedure (WO0238756) that uses low fidelity humanDNA polymerases (mutases) to introduce random mutations homogeneouslyover the whole target sequence. Three distinct mutases (pol β, pol η andpol ι) were used in different conditions to create complementarymutational patterns. These human polymerases were produced and purifiedas described previously (Mondon et al., Biotechnol J. 2: 76-82 (2007),Emond et al. Protein Eng. Des. Sel. 21: 267-274 (2008)).

I.2-a. Mutagenesis with the Mutagen™ Process

The human Fc gene (Fc gene) was double replicated with mutases using the5′ primer MG_619: 5′-AGTACTGACTCTACCTAGGATCCTGCCCACCGTGC-3′ (SEQ ID No5) and the 3′ primer MG_621 5′-ACTGCTCGATGTCCGTACTATGCGGCCGCGAATTC-3′(SEQ ID No 6) (BamHI and EcoRI restriction sites are underlined anditalic characters correspond to the non-specific tails). A mixturecontaining 0.6 μg of the pMG58_Fc226 plasmid as template (wild type Fcfor Mut1 library or Fc variants for Mut2 library), primers MG_619 andMG_621 (250 nM each) and the appropriate replication buffer (detailedbelow) was treated for 5 min. at 95° C. and immediately cooled down to4° C. to denature DNA strands. For pol β, replication buffer was 50 mMTris HCl pH 8.8, 10 mM MgCl₂, 10 mM KCl, 1 mM DTT and 1% (v/v) glycerol.Replication buffer for pol η (or pol η and pol ι) was 25 mM Tris HCl pH7.2, 5 mM MgCl₂, 10 mM KCl, 1 mM DTT and 2.5% (v/v) glycerol. After thedenaturation step, mutagenic replications were performed by adding 50 μMdATP/dCTP, 100 μM dTTP/dGTP and 1 μg of pol β or 100 μM dNTPs and 1 μgof pol η (or pol η and pol ι, 1 μg of each mutase). The replicationreaction was carried out at 37° C. for two hours. The replicationproducts were then desalted and concentrated on microcon columns(Millipore).

1.2.b. Selective Amplification and Cloning of Mutated Fragments

The replication products previously obtained were amplified through aselective PCR with tail primers. The primers (MG_619 and MG_621) weredesigned with a tail that is non-complementary to the template allowingspecific amplification of the DNA fragments synthesised by the mutases.A fraction of the replication products was added to a mixture containingthe PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl₂, 10 pmolof the 5′ and 3′ primers, 200 μM dNTPs and 1.25 U Platinum Taq DNApolymerase (InvitroGen). The PCR cycles were as follow, first cycle: 2min. at 94° C., 10 sec. at 64° C., 30 sec. at 75° C., 1 min at 94° C.and then 30 selective cycles: 20 sec. at 94° C. and 30 sec. at 75° C.

The amplified replication products were purified on 1% (w/v) agarosegels, digested with BamHI and EcoRI restriction enzymes and cloned intothe pMG58 vector. The ligation mixtures were transformed inelectrocompetent E. coli XL1-Blue cells and subsequently plated on solid2YT medium (16 g/l peptones, 10 g/l yeast extract, 5 g/l NaCl, 15 g/lagar) supplemented with 100 μg/ml ampicillin and 1% (w/v) glucose. Aftergrowth, the number of colonies was determined to estimate the size ofthe libraries and 96 clones per library were randomly subjected to PCRand high throughput DNA sequencing. Cells were scrapped in 2YT mediumwith 15% glycerol, frozen and kept at −80° C.

For the first round of mutagenesis and screening (MS1), four differentlibraries were constructed. A first library was obtained using pol β onthe wild type Fc gene and contained 3.2×10⁶ clones (called Mut1.1). TheDNA of this first library was used to generate the second and the thirdlibraries, using respectively pol β (3.8×10⁶ clones, Mut1.2) and pol ηand ι (3.0×10⁶ clones, Mut1.3). This strategy in two cumulativereplication steps permitted to increase the mutation rate. The fourthlibrary was generated with polymerase η alone on the wild type Fc gene(1.0×10⁶ clones, Mut1.4). Finally, these four libraries wereproportionally mixed to obtain the final library called Mut1,representing 1.1×10⁷ different clones.

For the second round of mutagenesis and screening (MS2), two differentlibraries were constructed using a DNA pool of 42 single and doublemutants isolated during MS1 and having improved FcRn-binding byphage-ELISA. A first library was obtained using pol β (1.9×10⁷ clones,Mut2.1) and a second library with pol η (1×10⁶ clones, Mut2.2). Finally,these two libraries were proportionally mixed to obtain the finallibrary called Mut2, representing 2×10⁷ different clones.

I.2.c. Quality Control of the Fc Libraries by Sequencing

The quality of the different libraries generated previously was assessedby PCR on cells to amplify the Fc gene (with the 5′ primer5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO: 7) and the 3′ primer5′-TCACGTGCAAAAGCAGCGGC-3′ (SEQ ID NO:8) and high throughput sequencing(with the 5′ primer 5′-TGATTACGCCAAGCTTGC-3′ (SEQ ID NO:9). Thesequences of 96 clones randomly picked in each library (Mut1.1 to Mut1.4and Mut2.1 to Mut2.2) were thereby determined. Finally, 35 clones of thepooled library Mut1 and 86 clones of the pooled library Mut2 were alsosequenced to control the quality of the final library before theselection process.

The modifications of the mutated sequences were analysed using MilleGenproprietary software Mutanalyse4Fc adapted for the Fc molecule from theMutanalyse 2.5 software described previously (Mondon et al., BiotechnolJ. 2: 76-82 (2007)). This analysis confirmed that the mutations arerandomly distributed along the entire gene, without any “hot spot”

Mut1 Analysis:

the frequency of mutations of Mut1 library is of 6.3 mutations per kilobases (kb), which means 4.2 mutations per gene (666 nt). Amongst thesemutations, 81.4% are substitutions, 16.8% are deletions and 1.8%additions, these last two categories introducing frame shifts in thegene. When considering only the sequences in frame, the mutationfrequency is of 4.0 mutations per kb, i.e. 2.7 mutations per gene (1 to6 mutated nucleotides per gene). The mutation analysis was alsoperformed at the protein level to determine the active part of thelibrary. Finally, the Mut1 library contains 28.6% of clones expressingthe wild type fragment (non mutated or with silent mutations), 40.0% ofclones containing a sequence out of frame or with a stop codon (notexpressed) and 31.4% of clones with a mutated sequence (Fc variants).These last clones represent the active part of the library, comprising3.5×10⁶ different clones with on average 2.3 mutated amino acids permolecule.

Mut2 Analysis:

the frequency of mutations of Mut2 library is of 4.5 mutations per kilobases (kb), which means 3.0 mutations per gene. Amongst these mutations,96.3% are substitutions, 3.2% are deletions and 0.5% additions. Whenconsidering only the sequences in frame, the mutation frequency is of4.3 mutations per kb, i.e. 2.9 mutations per gene (1 to 7 mutatednucleotides per gene). At the protein level, the Mut2 library contains17.4% of clones expressing the wild type fragment (non mutated or withsilent mutations), 9.3% of clones containing a sequence out of frame orwith a stop codon (not expressed) and 73.3% of clones with a mutatedsequence (Fc variants). These last clones represent the active part ofthe library, comprising 1.5×10⁷ different clones with on average 1.9mutated aa per molecule.

II. Phage Display Expression of the Fc Libraries and Selection of FcRnImproved Binders

The Fc library was expressed at the surface of the bacteriophage M13using standard procedures (Smith G P, Science 228: 1315 (1985)). E. coliXL1-Blue bacteria containing the Mut1 library (pMG58 vector) were grownin 60 ml of 2YT supplemented with 100 μg/ml ampicillin, 15 μg/mltetracycline and 1% (w/v) glucose at 30° C., 230 rpm untilOD_(600nm)=0.6 is reached. Cells were then infected with M13 helperphage (M13KO7, Biolabs, ratio bacteria/phage=1/3) at 37° C. for 20 minand phage-Fc production was continued overnight at 26° C., 230 rpm in2YT/Ampicillin/Glucose with IPTG 0.5 mM and kanamycin 30 μg/ml. Thefollowing day, phages were precipitated with PEG6000 using standardprotocols, resuspended in 1 ml phosphate buffer pH6 (sodium phosphate100 mM, sodium chloride 50 mM pH6.0, called P6) and titrated byinfecting XL1-Blue cells. Three selection strategies were applied usingdifferent conditions (FIG. 2) and 3 to 8 rounds of selection wereperformed per strategy (see below).

II.1. Selections on Solid Phase (Strategies 1 and 2) (See FIG. 2A):

For solid phase selections, 4×10¹¹ phages in P6/5% skimmed milk/0 1%Tween 20 were incubated on 8 wells of Maxisorp immunoplates previouslycoated with 0.5 μg neutravidin and 0.5 μg biotinylated FcRn (strategy 1)or 0.5 μg FcRn-p3 (strategy 2) and blocked with 5% skimmed milk in P6.After incubation for 2 hours at 37° C., wells were washed 20 times withP6/0.1% Tween 20 and eluted by incubation in 100 μl phosphate bufferpH7.4 (sodium phosphate 100 mM, sodium chloride 50 mM pH7.4)/well for 2hours at 37° C. After titration, eluted phages were used to reinfect 10ml of exponentially growing XL1-Blue bacteria and subsequently plated onsolid 2YT medium/ampicillin/glucose. The following day, cells werescrapped in 2YT medium with 15% glycerol, frozen and kept at −80° C.until the next round of selection.

II.2. Selection in Liquid Phase (Strategy 3) (See FIG. 2B):

For liquid phase selection, 4×10¹¹ phages were first incubated with 250nM or 100 nM biotinylated FcRn in 1 ml P6/5% skimmed milk/0.1% Tween 20for 1 hour at room temperature under low agitation. Streptavidin coatedmagnetic beads (Dynal), previously blocked with 5% skimmed milk in P6were then added to the phages for 30 minutes at room temperature.Phage-bead complexes were washed 15 times with P6/0.1% Tween 20 using amagnet (magnetic particle concentrator, Dynal). Phages were eluted byincubation in 500 μl phosphate buffer pH7.4 (sodium phosphate 100 mM,sodium chloride 50 mM, pH 7.4) for 2 hours at room temperature. Beadswere discarded using the magnet and eluted phages in the supernatantswere collected. After titration, eluted phages were used to reinfect 10ml of exponentially growing XL1-Blue bacteria and subsequently plated onsolid 2YT medium/ampicillin/glucose. The following day, cells werescrapped in 2YT medium with 15% glycerol, frozen and kept at −80° C.until the next round of selection.

II.3. Sequence Analysis:

During screening processes (MS1 and MS2), for each strategy, from round3 to round 8, 48 to 96 clones were sequenced after PCR on cells (asdescribed in I.2-c.). Sequence analysis was performed using MilleGenproprietary software AnalyseFc internally developed to rapidly analysethe selected Fc variants. Fc Variants were named according to the roundof selection from which they were isolated (for Mut1 screening: B3A toB6A for strategy 1, S3A to S6A for strategy 2 and L3A/B to L6A-F forstrategy 3, for Mut2 screening: C3A to C8A for strategy 1, T3A to T8Afor strategy 2 and M3A/B for strategy 3). Numbers (1 to 96) refer to thelocalisation on the PCR plate for sequencing. Finally during the wholeselection process, 227 different mutated clones were isolated for Mut1and 223 different mutated clones for Mut2. All these clones werecharacterised using phage-ELISA assays.

II.4. Directed Mutagenesis

The sequence analysis of the improved Fc-variants isolated during MS1showed that a large number of clones contained similar mutations (N434Y,N434S, P230S, P230T . . . ). Directed mutagenesis was performed toremove these mutations in order to reveal the effect of the associatedmutations. These new mutants were named based on the parental clone withan A or B added at the end of the name. 61 new mutants were tested. Someof these mutants are illustrated in table 1. The mutants considered aspositive have a specific signal between 1.2 and 2.6-fold higher thanthat of Fc-WT in phage ELISA assay (see below). After MS2, several newmutants were constructed by directed mutagenesis by adding one or twomutations in the hinge region (P230S or P228L or P228R or P228L/P230S orP228R/P230S). These mutants were named based on the parental clone witha letter (A to G) added at the end of the name. 24 new mutants weretested and are illustrated in table 5.

II.5. Phage-ELISA Assays of the Selected Variants (FIG. 3)

The binding characteristics of the variants isolated during MS1 and MS2displayed on the phage were determined using an ELISA test at pH6.0 withFcRn-p3 coated on wells (FIG. 3). Briefly, phage-Fc variants wereproduced as isolated clones on a 96-well plate in 800 μl cultures in2YT/ampicillin/glucose infected with helper phage M13K07 (as describedin paragraph 3). Phages produced overnight at 26° C. were recovered inthe supernatants after 30 minutes centrifugation at 3000 g. Thesesupernatants were directly diluted (1/2 and 1/4) in P6/5% skimmedmilk/0.1% Tween 20 and tested on Maxisorp immunoplates previously coatedwith 0.25 μg FcRn-p3/well and blocked with 5% skimmed milk in P6. Afterincubation for 2 hours at 37° C., wells were washed 3 times with P6/0.1%Tween-20 and bound phages were detected with an HRP anti-M13 antibody(GE Healthcare).

Using this ELISA test, the 227 Fc variants selected during MS1 weretested in comparison with the wild type Fc (Fc-WT) and a positivecontrol. This positive control (called Fc-H variant) is the doublemutant T250Q/M428L and was described as having an improved affinity forFcRn (×28, Hinton P R et al., J. Biol. Chem. 279(8): 6213-6216 (2004)).This variant was generated by standard PCR protocols with two longoligonucleotides comprising the mutated codons and the restrictionsites: 5′ primer

(SEQ ID NO: 10) 5′-CGGGATCCTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACCAACTCATGATCTCCCG GAC-3′and 3′ primer

(SEQ ID NO: 11) 5′-GCGAATTCTTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGAGCCTCATGCAGCACGGAGCATGAGAAG-3′(BamHI and EcoRI restriction sites are underlined and the characters ingrey correspond to the mutated codons). In the phage-ELISA assay, theFc-H variant had a specific signal on average 3.2-fold stronger than thewild type Fc, i.e. Fc-WT (ratio variant/Fc-WT) and amongst the 227Fc-variants tested, 73 variants had a ratio/Fc-WT>3.2, which means thatthey had a better binding to FcRn than the Fc-H variant (table 2).Positive variants from MS1 and having a single point amino acidmodification have a specific signal from about 1.2-fold to 3.5-foldstronger than the wild-type Fc (see table 1).

TABLE 1 Variants having a single amino acid modification identifiedduring MS1 or obtained by directed mutagenesis Variant name Mutationratio/Fc-WT B4A_13 P228L 3.5 B3A_32 P228R 3.1 B5A_35 P230S 2.8 L3A_20V303A 2.8 L5D_47 P230Q 2.7 S3A_05 N434S 2.7 B4A_22A A378V 2.6 B5A_05H433R 2.3 S3A_04 P230T 2.3 B4A_08 V397M 2.2 B5A_25B N315D 2.1 B5A_15M428L 2.0 L3B_21A V302A 1.9 S3A_25A V264E 1.9 L3A_01 T256N 1.8 S3A_08P387S 1.8 L3A_35 S440N 1.7 L3B_20 E382G 1.7 B3A_08 C226G 1.6 B5A_17AQ362E 1.6 B5A_43 R416G 1.6 L5A_01 N389K 1.5 S3A_09A S426T 1.4 S3A_21N297D 1.4 B3A_17 T307A 1.3 B5A_31A Q342R 1.2 L3A_10 L309P 1.2 L3A_16A378T 1.2 L3A_25 V264A 1.2 L3B_19 Q386R 1.2 B4A_12 K414R 1.1 B4A_29K447E 1.1 L4F_02 A330T 1.1 L5D_01 V305A 1.1 L6A_39 N389T 1.1 S5A_18AF404L 1.1 L5D_29A Q342K 1.1 B4A_44A K290R 1.1 L6C_10B D265G 1.1 L4A_39D401G 1.0 B6A_34A N390S 1.0 L6C_44A T359A 1.0 B5A_04A N384I 1.0 S3A_24AE269D 1.0 L5D_09A T289I 1.0 S3A_01 Q311R 0.9 S3A_42 K360R 0.9 L4F_14G371D 0.9 L5B_35 N276S 0.9 L6A_29 S267N 0.9 B3A_02A N421S 0.9 S4A_17BQ362R 0.9 S3A_26A T394A 0.9 B5A_14A Q347R 0.9 L6B_22 P395S 0.8 B5A_28AK360N 0.8 L5D_41A K322R 0.8 L5A_31 N361S 0.5

TABLE 2 variants selected during MS1 Variant name Mutations Ratio/Fc-WTS5A_41 P230T/V303A/K322R/N389T/F404L/N434S 9.0 B5A_01 P228R/N434S 8.2S5A_26 Q311R/K334R/Q342E/N434Y 7.9 B4A_21 C226G/Q386R/N434Y 6.9 S4A_07T307P/N389T/N434Y 6.6 B5A_48 P230S/N434S 6.5 L6B_31P230T/V305A/T307A/A378V/L398P/N434S 6.3 S4A_01 P230T/P387S/N434S 6.2S3A_24 P230Q/E269D/N434S 6.1 S4A_14 N276S/A378V/N434S 5.9 S4A_12R355Q/T393N/S426T/N434Y 5.8 S5A_47 P230T/N434S 5.8 S5A_43P230S/V284L/A378V 5.7 B5A_16 S239A/S298G/N315D/Q347R/N434Y/S440R 5.6B5A_23 Q362E/N434Y 5.4 S4A_24 V264E/R301C/A378V/E382G 5.4 L4A_28M428L/N434S/Q438R/P445S 5.2 S4A_03 A378V/N434S 5.2 S4A_29P230Q/F241L/V264E 5.2 S5A_07 A378V/N421T/N434S 5.1 B6A_31S375G/M428L/H433P 5.0 L5D_09 P230Q/T289I/N434S 4.8 S4A_06K288R/T307P/N421S/N434S 4.7 B5A_17 N315D/Q362R/N434Y 4.6 S4A_02A378V/N434Y 4.6 S4A_36 P230Q/V264A/P352S/A378V 4.5 S4A_44 P227S/N434Y4.5 L5B_14 C226G/N434S 4.4 L6B_41 P230S/M428L 4.4 S6A_48R355Q/K392E/T393N/S426T/N434Y 4.4 B6A_41 N434Y/Q438R/K447E 4.3 L5D_18V397M/N434S 4.3 S3A_25 V264E/N434S 4.3 S5A_20 P230Q/F2 41Y/K246R/D270E4.3 S4A_25 D265G/S408T/N434Y/S444F 4.2 S4A_42 V264A/N434Y/G446A 4.2B5A_18 V412A/M428L/H433R/N434S/K447E 4.1 B4A_39 E382G/N434S 4.0 L4A_45P228L/N297D 4.0 L6A_11 M428L/H433R 4.0 S4A_30 V303A/N434S 4.0 B4A_01E345Q/A378S/E380Q/N434Y 3.9 B4A_22 S383R/V397M/N434S 3.9 S4A_17V302A/N389T/N434S 3.8 B4A_03 R292W/T307P/A330V/N434S 3.7 B5A_04N384I/N434Y 3.7 B6A_20 K320T/N434Y/K439R/K447E 3.7 S4A_05A378V/D401A/N434Y 3.7 S4A_11 N389T/N434Y 3.7 S6A_24 A231T/V397M/N434S3.7 B4A_46 G371D/N434Y 3.6 B5A_25 F243L/N315D/T411A/N434S 3.6 S3A_06P230T/A231T/A378V 3.6 B3A_02 N421S/N434Y 3.5 B4A_13 P228L 3.5 S3A_07V264E/A378V/E382G 3.5 S4A_27 M252L/N434S 3.5 B3A_15 L309P/N434S 3.4B3A_34 N434Y/S440N 3.4 B6A_34 N315D/A330V/N434S 3.4 S3A_09S426T/N434S/K439R 3.4 S5A_46 Q386R/N434Y 3.4 B4A_44 P230S/K29OR 3.3B6A_36 F241L/V305A/D356N/N434Y 3.3 L5D_29 Q342K/N434Y 3.3 S5A_05N434Y/S440R 3.3 S5A_19 F243L/N434Y 3.3 S5A_27 A327V/A378V/N389T/N434Y3.3 B5A_28 K360N/N434Y 3.2 L3A_39 S375G/P395S/N434S 3.2 L4A_15P395S/N434H 3.2 L4F_16 N434Y/K447N 3.2 S5A_40 T299M/N434Y 3.2

The 223 variants selected during MS2 were tested using the same ELISAprotocol but in comparison with the Fc-H variant and the best Fc variantisolated during MS1 (S5A_41), because the difference between the Fc-WTsignal and the signal of the Fc variants was too great to be compared onthe same ELISA plate. Amongst the 223 Fc variants tested, 209 Fcvariants were better than the Fc-H (ratio/Fc-H>1.1) and 39 Fc variantwere better than the best Fc variant isolated during MS1. To compare thevariants with the Fc-WT, an estimated ratio/Fc-WT was calculated bymultiplying the ratio/Fc-H of the variants by the ratio/Fc-WT of theFc-H (=3.2) determined during MS1 (ratio/Fc-WT=3.2×ratio/Fc-H) (table 3)

TABLE 3 variants of MS2 Variant Ratio/ Ratio/ name Mutations Fc-H FWTC6A_69 T307A/N315D/A330V/E382V/N389T/N434Y 8.9 28.4 C6A_78T256N/A378V/S383N/N434Y 8.7 27.8 T5A_74 N315D/A330V/N361D/A378V/N434Y8.6 27.6 C6A_74 V259I/N315D/N434Y 8.5 27.2 C6A_60P230S/N315D/M428L/N434Y 8.4 26.8 T5A_58 F241L/V264E/T307P/A378V/H433R8.1 26.1 C6A_72 T250A/N389K/N434Y 8.0 25.7 T5A_93V305A/N315D/A330V/P395A/N434Y 8.0 25.7 T5A_78V264E/Q386R/P396L/N434S/K439R 8.0 25.6 T5A_87 N315D/A330V/Q362R/N434Y7.8 25.0 C6A_66 E294del/T307P/N434Y 7.7 24.6 C6A_85V305A/N315D/A330V/N389K/N434Y 7.4 23.8 C8A_15N315D/A327V/A330V/V397M/N434Y 7.4 23.7 T5A_89P230T/F241L/V264E/D265G/A378V/N421T 7.1 22.8 T7A_92V264E/P396L/S415N/N434S 6.7 21.4 T6A_57 P227L/V264E/A378V/N434S 6, 4 fhr20.3 6.4 T5A_94 V264E/A378T/P396L 5.8 18.5 T6A_75P230T/N315D/Q362R/S426T/N434Y 5.7 18.3 C3A_13 C226G/N315D/A330V/N434Y5.6 17.9 T5A_55 P230L/F241L/F243L/V264E/T307P/A378V 5.6 17.9 T6A_85T250A/N315D/N325S/A330V/N434Y 5.1 16.3 C5A_39K290E/N315D/Q342R/E382V/N434Y 5.0 15.9 T5A_57F241L/N315D/A330V/K392R/N434Y 4.9 15.8 C5A_09F241L/V264E/T307P/A378V/N434S 4.8 15.2 T6A_22 P230T/V264E/S403T/N434S4.7 15.2 T5A_81 V264E/A378V/R416K 4.6 14.9 C6A_12P230T/N315D/Q362E/N434Y 4.6 14.9 C4A_14 C226G/N315D/N434Y 4.6 14.8T4A_42 C226G/N315D/Q362R/N434Y 4.6 14.7 T5A_25C226G/V264E/Q347R/K370R/A378V/N434S 4.6 14.7 T4A_48V308I/N315D/A330V/E382V/N434Y 4.5 14.5 C6A_48 P230T/V264E/A378V/N434S4.5 14.4 T5A_45 A231T/F241L/V264E/A378T/V397M/N434S 4.5 14.3 T6A_23P230L/V264E/A378V/N434S 4.4 14.1 C5A_65 P230T/N315D/A330V/Q386K/N434Y4.2 13.5 C6A_88 C226G/N315D/A330V/N389T/N434Y 4.2 13.4 C4A_13S267R/T307P/A378V/N421T/N434Y 4.1 13.2 C3A_35 P230S/N315D/P387T/N434Y4.0 12.9 T4A_37 P230S/V264E/P352S/A378V/N434S 4.0 12.8 C5A_18P230T/N315D/Q362R/N434Y 3.9 12.3 T3A_22 F241L/V264E/A378V/N434S 3.8 12.2C5A_12 N315D/Q362E/N389K/N434Y 3.8 12.1 C4A_29T307P/N315D/N361S/Q362R/N434Y 3.8 12.0 T4A_44 C226G/V264E/A378V/F404L3.8 12.0 C3A_42 N315D/A330V/N389K/V397M/N434Y 3.7 12.0 C7A_82P230T/K246R/N389T/P395S/N434Y 3.7 11.9 T4A_31P230T/F241L/V264E/T307P/A378V 3.7 11.7 T7A_48P230T/L234R/N315D/A330V/N434Y 3.6 11.6 T7A_49P230T/N315D/K320E/Q362R/N434Y 3.6 11.6 C7A_43V264E/T307P/A378V/P396S/N434S 3.6 11.5 T4A_26V264E/T307P/A378V/E382G/Q386R 3.6 11.5 T4A_19 T307P/A378V/N434S 3.6 11.4C4A_06 P230T/N315D/A330V/N434Y 3.6 11.4 T4A_46 P230T/N389K/N434Y 3.611.4 T8A_24 V264E/T307P/A378V/N434S 3.5 11.1 C6A_36T307P/N315D/E382G/Q419H/N434Y 3.4 10.9 C7A_68V264E/N315D/A378V/N390S/G420R/N434Y 3.4 10.9 C5A_15V303A/N315D/A330V/E382V/N434Y 3.4 10.9 T5A_40P230T/V264E/T307P/A378V/N421T 3.4 10.8 C4A_28 V264E/A378V/N434Y 3.4 10.8C4A_41 N315D/A330V/Q362E/N434Y 3.4 10.8 T6A_42 C226G/N434Y 3.4 10.7T4A_33 P230T/V264E/A378V/N389T/D399N/H433R 3.3 10.7 T5A_24V264E/A378V/N434S 3.3 10.7 T8A_87 F241L/V264E/A378V/N421T/N434S/L443R3.3 10.7 T6A_39 C226Y/A378V/N421T/N434S 3.3 10.4 C3A_45F243L/N315D/A330V/N389K/N434Y 3.3 10.4 C3A_09 S298G/N434Y 3.2 10.4T6A_21 N315D/A378V/N434Y 3.1 10.0 C6A_13 T307P/A378V/N434Y 3.1 10.0C3A_27 N315D/S354P/S383N/N434Y 3.1 10.0 T6A_16P230T/V264E/N315D/K370R/A378V 3.1 9.9 C3A_21 N315D/A330V/S400P/N434Y 3.09.8 C3A_08 V264E/P352S/A378V/N434S 3.0 9.7 T4A_18N315D/Q342R/E382V/N434Y 3.0 9.7 T4A_04 N315D/A330V/E382V/N434Y 3.0 9.7T7A_58 N315D/Q362E/N434Y 2.9 9.4 C6A_04 Q342R/E382V/N434Y 2.9 9.4 C5A_19V264A/V305A/N315D/A330V/N434Y 2.9 9.3 T6A_13P230T/N315D/A330V/Q362R/N434Y 2.9 9.3 T7A_87 P230S/N315D/Q362R/N434Y 2.99.3 C3A_24 T307P/N389T/D401G/N421T/N434Y 2.9 9.2 C4A_22 P230T/N434Y 2.99.1 T6A_47 P230T/K320T/N434Y 2.8 9.1 C5A_58 V264E/A378V/P396L/N434S 2.89.1 T6A_40 P230A/F241L/V264E/A378V/N421T 2.8 9.1 T5A_51V264E/A378V/F404L/N434S 2.8 9.0 C4A_25 N315D/A330V/V397M/N434Y 2.8 8.9T3A_15 V264E/A378V/T394A/F404L/N434S 2.8 8.9 C7A_18V264E/A378V/K414R/N421T/N434Y 2.8 8.9 T7A_18 V264E/A378V/Q386R/N434S 2.88.9 C4A_36 N315D/K320T/N434Y 2.8 8.9 C5A_75 T307N/N315D/N434Y 2.8 8.9C5A_28 T307P/N434Y 2.8 8.8 T5A_05 V264E/E269G/A378V/N421T/N434S 2.7 8.8C8A_41 N315D/E382V/H433P/N434Y 2.7 8.8 C5A_44 N315D/N389K/N434Y 2.7 8.7C5A_03 V264A/N315D/N434Y 2.7 8.7 T4A_45 V264E/L309P/P396L/N434S 2.7 8.7C4A_27 V264A/T299A/A378V/E382G/N434Y 2.7 8.7 T6A_12V264E/A378V/N421T/N434S 2.7 8.6 T7A_76 V264E/K370R/A378V/P396L/H439R 2.78.5 T5A_08 V264E/P291Q/A378V/N434S 2.6 8.5 M3A_21F241L/V264E/T307P/A378V/N421T 2.6 8.4 C5A_20 N315D/S415D/N434Y 2.6 8.4T6A_09 P230T/T307A/N315D/A327V/N434Y 2.6 8.3 C7A_27V264E/T307N/A378V/V397M/N434Y 2.6 8.2 T7A_46 V264E/A378V/G385R/N434S 2.68.2 T8A_81 V264A/N315D/E382V/N434Y 2.5 8.2 T7A_57P230T/T307A/N315D/A330V/Q418E/N434Y 2.5 8.1 C3A_43 N315D/R416G/N434Y 2.58.1 C4A_18 N315D/A330V/A378V/N434Y 2.5 8.1 T4A_41F241R/V264E/T307P/A378V 2.5 8.0 C3A_01 N315D/A330V/N434Y 2.5 8.0 T8A_41V264E/P343S/A378V/N434S 2.5 7.9 T5A_28 F241L/V264E/A378V/N434Y 2.5 7.9T3A_10 N315D/A330V/N389K/N434Y 2.4 7.8 T3A_01 V264E/T307P/A378V/N421T2.4 7.8 T5A_59 Q342R/R355G/E382V/N434Y 2.4 7.8 M3B_09V264A/N315D/A330V/N389K/N434Y 2.4 7.7 C8A_14 V305A/Q386R/N434Y 2.4 7.6C4A_01 N315D/Q362R/N389K/N434Y 2.4 7.5 C4A_24 L309P/N315D/A330V/N434Y2.3 7.5 C7A_13 F241L/V264E/A378V/N421T/N434Y 2.3 7.4 T4A_32V264E/A378V/H433R 2.3 7.4 T3A_16 F241L/V264E/T307P/A378V 2.3 7.4 C7A_89T307P/A327T/N389T/N421T/N434Y 2.3 7.4 C5A_50 D270N/N315D/N434Y 2.3 7.3T3A_41 V264E/T307P/A378V 2.3 7.2 C4A_45 K246R/H285Y/N315D/A330V/N434Y2.3 7.2 T7A_24 V264E/A378V/N421T/N434Y 2.3 7.2 T4A_28 P230T/A378V 2.27.0 T5A_37 S298G/N315D/A330V/N434Y 2.2 7.0 C3A_31N315D/A330V/N389K/D401G/N434Y 2.2 6.9 C4A_15 E233D/N315D/N434Y 2.2 6.9C7A_02 V264E/K370R/A378V/N434Y 2.2 6.9 C7A_37 F241L/N315D/N389K/N434Y2.2 6.9 C7A_69 V264E/H285Y/A378V/N434Y 2.1 6.9 C7A_52 D265G/A378V/N434Y2.1 6.8 T5A_64 P230T/V264A/N325S/V397M/N434S 2.1 6.7 C7A_23S298N/A378V/N434Y 2.1 6.6 C7A_67 N315D/A330V/N389R/N434Y 2.1 6.6 T5A_03V264E/P396L/N434S 2.0 6.4 C8A_08 V264A/N315D/A330V/N434Y 2.0 6.4 C3A_03N315D/N434Y 2.0 6.4 T3A_47 T307P/A378T/V397M 2.0 6.3 C5A_63S298N/N315D/A330V/N434Y 2.0 6.3 T7A_17 V264E/P291S/Q362R/A378V/N434Y 2.06.3 T4A_43 1332V/K370R/A378V/N434S 2.0 6.3 M3A_18 T307P/A378V/N421T 1.96.2 C6A_05 T307A/N315D/A330V/N434Y 1.9 6.2 C3A_15 V264E/T307A/A378V 1.96.0 T5A_29 N315D/E382V/N434Y 1.9 6.0 T5A_52 N315D/A327V/A330V/N434Y 1.95.9 T8A_45 S375A/A378V/N434Y 1.8 5.9 C6A_21N315D/A330V/K360R/N389K/N434Y 1.8 5.8 T7A_05 V264E/T359A/N434Y 1.8 5.8T8A_50 V264E/P396L/N434Y 1.8 5.8 T7A_94 S267N/P352S/A378V/P396L/N434S1.8 5.8 C6A_35 T250A/N315D/A330V/N434Y 1.8 5.7 C7A_22N315D/K334E/A378V/N434Y 1.8 5.7 M3A_06 F241L/V264E/A378T/V397M 1.8 5.7T7A_13 C226Y/N315D/N434Y 1.8 5.7 T5A_90 N315D/A330V/K392R/S424L/N434Y1.8 5.7 C3A_39 A231V/Q342E/N434Y 1.8 5.6 T3A_13 N315D/V369A/N434Y 1.85.6 T8A_34 T307A/N315D/T335A/N434Y 1.8 5.6 M3A_26V264E/T307P/K340E/Q342R/A378V 1.8 5.6 C3A_23 N389K/N434Y 1.8 5.6 M3A_08V264E/T307P/A378T/V397M 1.7 5.6 C8A_61 P230T/V264E/P396L/N434Y 1.7 5.5M3B_04 F241L/V264E/Q342R/A378V 1.7 5.5 C4A_32 V264E/N315D/A378V 1.7 5.4T7A_35 N315D/Q362R/N434Y/S444P 1.7 5.4 C7A_49 N315D/A330V/T394A/N434Y1.7 5.3 C7A_28 N315D/S383N/N434Y 1.6 5.3 T6A_58F241L/V264E/T307P/K338R/A378V/N434S 1.6 5.2 C6A_33 S426T/N434Y 1.6 5.2C6A_93 V264M/D265N/N315D/A330V/N434Y 1.6 5.0 M3A_22 P230S/A378V/K439R1.5 5.0 T3A_37 F241L/V264E/A378V 1.5 4.9 T7A_95 N315D/Q342R/N384T/N434Y1.5 4.9 C3A_18 F241L/V264E/A378V/N421T 1.5 4.8 T3A_28 T307P/A378V/Q418R1.5 4.8 T3A_06 T307P/A378V 1.5 4.8 C6A_23 V264E/N434Y 1.5 4.7 T3A_21N315D/K317R/N434Y 1.5 4.7 T3A_34 V264E/P352S/A378V 1.4 4.6 C5A_48T350A/N434Y 1.4 4.6 T3A_43 V264E/E345G/A378V 1.4 4.5 M3A_01 N361D/N434Y1.4 4.4 T4A_39 V264E/A378V/P396L 1.4 4.4 C5A_41N315D/A327T/A330V/Q362R/N434Y 1.3 4.3 M3A_34 S267N/T307N/K370R/A378V 1.34.3 T4A_34 V264E/A378V/Q418K 1.3 4.3 C3A_07 T307P/A330T/A378V 1.3 4.2T3A_11 P291S/N315D/A327V/A330V/N434Y 1.3 4.0 C6A_02T307N/N315D/A330V/N434Y 1.2 3.9 T3A_09 V264E/A378V/N421T 1.2 3.9 T5A_44F241L/V264E/T307P/A378T/V397M 1.2 3.8 T3A_12 T256N/A378V 1.2 3.8 M3B_23F241L/V264E/T307P 1.2 3.8 M3A_35 V264E/N315D/P396L 1.1 3.7 T3A_26V397A/N434Y 1.1 3.6 T3A_08 V264E/A378V/F404L 1.1 3.5 T6A_93T299K/Q311R/N315D/N434Y 1.1 3.5 M3A_12 N315D/Q362R/N421D/N434S 1.1 3.4T3A_20 V303I/N434Y 1.1 3.4 T3A_30 V264E/A378V/V422A 1.1 3.4

Overall, 282 Fc variants having better binding for FcRn than Fc-H wereisolated during MS1 and MS2 processes. Analysis of the sequences ofthese 282 Fc variants revealed that they include mutations all over themolecule on 115 different positions (table 4).

TABLE 4 mutations of MS1 and MS2 variants Position Percentage ofvariants Modification C226 3.9 G or Y P227 0.7 S or L P228 1.1 R or LP230 16.3 S, T, L, A or Q A231 1.4 T or V E233 0.4 D L234 0.4 R S239 0.4A F241 9.2 L, Y or R F243 1.4 L K246 1.1 R T250 1.1 A M252 0.4 L T2560.7 N V259 0.4 I V264 33.0 A, E or M D265 1.4 G or N S267 1.1 N or RE269 0.7 D or G D270 0.7 N or E N276 0.4 S V284 0.4 L H285 0.7 Y K2880.4 R T289 0.4 I K290 0.7 R or E P291 1.1 S or Q R292 0.4 W E294 0.4deletion N297 0.4 D S298 1.8 G or N T299 1.1 M, A or K R301 0.4 C V3020.4 A V303 1.4 A or I V305 2.1 A T307 16.3 P, A or N V308 0.4 I L309 1.1P Q311 0.7 R N315 34.0 D K317 0.4 R K320 1.4 T or E K322 0.4 R N325 0.7S A327 2.5 V or T A330 17.0 V or T I332 0.4 V K334 0.7 E or R T335 0.4 AK338 0.4 R K340 0.4 E Q342 3.6 R, E or K P343 0.4 S E345 0.7 Q or G Q3470.7 R T350 0.4 A P352 1.8 S S354 0.4 P R355 1.1 Q or G D356 0.4 N T3590.4 A K360 0.7 N or R N361 1.1 D or S Q362 6.7 R or E V369 0.4 A K3702.1 R G371 0.4 D S375 1.1 A or G A378 37.2 V, T or S E380 0.4 Q E382 6.0V or G S383 1.4 R or N N384 0.7 I or T G385 0.4 R Q386 2.5 R or K P3870.7 S or T N389 9.2 T, K or R N390 0.4 S K392 1.1 E or R T393 0.7 N T3940.7 A P395 1.4 A or S P396 4.6 S or L V397 5.0 A or M L398 0.4 P D3990.4 N S400 0.4 P D401 1.1 A or G S403 0.4 T F404 1.8 L S408 0.4 T T4110.4 A V412 0.4 A K414 0.4 R S415 0.7 D or N R416 0.7 K or G Q418 1.1 R,K or E Q419 0.4 H G420 0.4 R N421 7.8 T, S or D V422 0.4 A S424 0.4 LS426 1.8 T M428 2.1 L H433 2.8 R or P N434 79.1 Y, S or H Q438 0.7 RK439 1.4 R S440 1.1 R or N L443 0.4 R S444 0.7 F or P P445 0.4 S G4460.4 A K447 1.4 E or N

Moreover, 16 positions are preferentially mutated and are considered askey positions: C226, P230, F241, V264, T307, N315, A330, Q342, Q362,A378, E382, N389, P396, V397, N421 and N434. Particularly, 4 positionsare more preferably mutated and are considered as most preferred keypositions: V264, N315, A378 and N434 (FIG. 4).

Fc variants of MS2 having better binding for FcRn compared to the bestvariant of MS1 (S5A_41), are showed in Table 5.

TABLE 5 best variants of MS2 Stan- Variant Ratio/ dard de- Ratio/ nameMutations Fc-H viation Fc-WT C6A_69 T307A/N315D/A330V/E382V/N389T/ 8.91.7 28.4 N434Y C6A_78 T256N/A378V/S383N/N434Y 8.7 1.9 27.8 T5A_74N315D/A330V/N361D/A378V/N434Y 8.6 1.6 27.6 C6A_74 V259I/N315D/N434Y 8.51.5 27.2 C6A_60 P230S/N315D/M428L/N434Y 8.4 1.8 26.8 T5A_58F241L/V264E/T307P/A378V/H433R 8.1 1.5 26.1 C6A_72 T250A/N389K/N434Y 8.01.1 25.7 T5A_93 V305A/N315D/A330V/P395A/N434Y 8.0 1.6 25.7 T5A_78V264E/Q386R/P396L/N434S/K439R 8.0 1.5 25.6 T5A_87N315D/A330V/Q362R/N434Y 7.8 1.4 25.0 C6A_66 E294del/T307P/N434Y 7.7 0.924.6 C6A_85 V305A/N315D/A330V/N389K/N434Y 7.4 1.5 23.8 C8A_15N315D/A327V/A330V/V397M/ 7.4 1.8 23.7 N434Y T5A_89P230T/F241L/V264E/D265G/A378V/ 7.1 1.2 22.8 N421T T7A_92V264E/P396L/S415N/N434S 6.7 1.5 21.4 T6A_57 P227L/V264E/A378V/N434S 6.41.7 20.3 T5A_94 V264E/A378T/P396L 5.8 1.0 18.5 T6A_75P230T/N315D/Q362R/S426T/N434Y 5.7 1.3 18.3 C3A_13C226G/N315D/A330V/N434Y 5.6 0.9 17.9 T5A_55P230L/F241L/F243L/V264E/T307P/ 5.6 1.2 17.9 A378V T6A_85T250A/N315D/N325S/A330V/N434Y 5.1 1.7 16.3 C5A_39K290E/N315D/Q342R/E382V/N434Y 5.0 0.6 15.9 T5A_57F241L/N315D/A330V/K392R/N434Y 4.9 1.0 15.8 C5A_09F241L/V264E/T307P/A378V/N434S 4.8 0.2 15.2 T6A_22P230T/V264E/S403T/N434S 4.7 0.9 15.2 T5A_81 V264E/A378V/R416K 4.6 1.014.9 C6A_12 P230T/N315D/Q362E/N434Y 4.6 0.6 14.9 C4A_14C226G/N315D/N434Y 4.6 0.8 14.8 T4A_42 C226G/N315D/Q362R/N434Y 4.6 0.414.7 T5A_25 C226G/V264E/Q347R/K370R/A378V/ 4.6 0.2 14.7 N4345 T4A_48V308I/N315D/A330V/E382V/N434Y 4.5 0.7 14.5 C6A_48P230T/V264E/A378V/N434S 4.5 0.8 14.4 T5A_45A231T/F241L/V264E/A378T/V397M/ 4.5 0.6 14.3 N4345 T6A_23P230L/V264E/A378V/N434S 4.4 0.7 14.1 C5A_65P230T/N315D/A330V/Q386K/N434Y 4.2 0.5 13.5 C6A_88C226G/N315D/A330V/N389T/N434Y 4.2 0.4 13.4 C4A_13S267R/T307P/A378V/N421T/N434Y 4.1 0.3 13.2 C3A_35P230S/N315D/P387T/N434Y 4.0 0.7 12.9 T4A_37P230S/V264E/P352S/A378V/N434S 4.0 0.5 12.8 55A_41P230T/V303A/K322R/N389T/F404L/ 3.9 0.6 12.4 N4345 C6A_78DP228R/T256N/A378V/N434Y 28.3 6.7 90.5 T5A_74DP228R/N315D/A330V/N361D/A378V/ 26.0 5.8 83.1 N434Y C6A_74DP228R/V259I/N315D/N434Y 18.6 4.6 59.7 T5A_74FP228R/P2305/N315D/A330V/N361D/ 16.8 5.5 53.8 A378V/N434Y C6A_78BP228L/T256N/A378V/N434Y 14.4 1.9 45.9 C6A_69GP228R/P2305/T307A/N315D/A330V/ 11.9 4.2 38.2 E382V/N389T/N434Y C6A_74CP228L/V259I/N315D/N434Y 11.0 2.8 35.3 C6A_69EP228R/T307A/N315D/A330V/E382V/ 10.0 2.8 32.0 N389T/N434Y C6A_78FP228R/P230S/T256N/A378V/N434Y 9.4 1.8 30.1 C6A_78CP230S/T256N/A378V/N434Y 9.2 2.4 29.4 C6A_78A T256N/A378V/N434Y 8.7 1.027.8 C6A_74E P228L/P230S/V259I/N315D/N434Y 8.6 2.7 27.5 C6A_60BP228R/N315D/M428L/N434Y 8.6 3.9 27.4 C6A_69CP230S/T307A/N315D/A330V/E382V/ 8.4 1.3 26.8 N389T/N434Y C6A_69FP228L/P230S/T307A/N315D/A330V/ 8.2 3.1 26.3 E382V/N389T/N434Y T5A_74CP228L/N315D/A330V/N361D/A378V/ 7.5 1.3 24.0 N434Y C6A_60DP228R/P230S/N315D/M428L/N434Y 7.4 1.4 23.8 C6A_74FP228R/P230S/V259I/N315D/N434Y 7.3 1.9 23.2 T5A_74EP228L/P230S/N315D/A330V/N361D/ 7.1 2.0 22.6 A378V/N434Y C6A_78EP228L/P230S/T256N/A378V/N434Y 6.0 0.4 19.1 C6A_74AP230S/V259I/N315D/N434Y 6.0 1.0 19.0 T5A_74BP230S/N315D/A330V/N361D/A378V/ 5.9 1.0 18.8 N434Y C6A_60CP228L/P230S/N315D/M428L/N434Y 5.3 1.9 17.1 C6A_69DP228L/T307A/N315D/A330V/E382V/ 4.8 1.0 15.3 N389T/N434Y C6A_60AP228L/N315D/M428L/N434Y 2.4 1.0 7.7

III. E. Coli Expression of the Fc Variants

The Fc-WT sequence as well as the Fc-H variant and Fc variants isolatedduring MS1 and MS2 were subcloned from the pMG58 phagemid vector intothe pMG62 vector, using BamHI and EcoRI restriction sites, permittingsoluble periplasmic expression with a C-terminal 6×His tag forpurification and a V5 tag for detection in ELISA assays. Production ofrecombinant Fc polypeptides was performed in HB2151 E. coli strain(induction with 0.5 mM IPTG for 16 hours at 20° C.). Purification wasperformed on Ni-NTA using standard protocols and around 200-504 μg ofeach polypeptide were obtained.

IV. FcRn Binding Characterisation of the Fc Variants Using ELISA andSurface Plasmon Resonance (SPR)

IV.1. FcRn Binding Characterization of S5A_41, S3A_07 and Fc-H variantsas compared to Fc_WT

IV.1.a. ELISA Assays

The binding characteristics of the Fc variants produced in a solubleformat were determined in comparison with the Fc-WT using an ELISA testat pH6.0 with FcRn-p3 coated on wells. Purified Fc variants (asdescribed in III) serially diluted in P6/5% skimmed milk/0.1% Tween-20were tested on Maxisorp immunoplates previously coated with 0.25 μgFcRn-p3/well and blocked with 5% skimmed milk in P6. After incubationfor 2 hours at 37° C., wells were washed 3 times with P6/0.1% Tween-20and bound Fc-variants were detected with an HRP anti-V5 antibody(invitroGen) (measurement of OD450 nm) (FIG. 5a). ELISA test wasperformed on S5A_41, the best variant of MS1, and S3A_07, a variantequivalent to Fc-H variant, and Fc-H variant. These ELISA testsconfirmed that Fc-H, S5A_41 and S3A_07 had improved binding to FcRncompared with the Fc-WT (FIG. 5b, FIG. 5c and FIG. 5d). For each bindingcurve, the measurement of Fc concentration at 50% saturation of thecurve (EC50) was used to characterise the binding properties of the Fcvariants compared to Fc-WT. The ratios thereby obtained confirmed thatthe S5A_41 is a better binder than Fc-H variant and S3A_07 is equivalentto Fc-H variant (Table 6).

IV.1.b. SPR (Surface Plasmon Resonance) Assays

The interaction of Fc variants with immobilized FcRn was monitoredperformed on a BIAcore X100 instrument using a CM5 sensor chip (Biacore,GE Healthcare). The methodology was similar to that previously describedfor analyzing Fc-FcRn interactions (Popov S. et al., Mol Immunol. 33(6): 521-530 (1996)). Recombinant soluble FcRn was coupled to flow cell2 of the sensor chip using amine-coupling chemistry. The flow cells wereactivated for 3 min with a 1:1 mixture of 0.1 M N-hydroxysuccinimide and0.1 M 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at a flow rate of30 μl/min. Recombinant human FcRn (5 μg/ml in 10 mM sodium acetate, pH5.0) was injected over flow cell 2 for 8 min at 10 μl/min, whichresulted in a surface density of 1200 to 1300 response units (RU).Surfaces were blocked with a 3-min injection of 1 M ethanolamine-HCl, pH8.5. Flow cell 1 was used as a control surface without FcRn and wasprepared similarly to sample flow cell. The data from this blank flowcell were subtracted from the sample data.

Fc fragments were diluted in PBS/Tween-20 (50 mM phosphate buffer, pH6.0, 150 mM NaCl, 0.02% NaN₃, 0.01% Tween-20) which is used as runningbuffer in equilibrium binding experiments. All measurements wereperformed at 25° C. with Fc fragment concentrations typically rangingfrom 1 to 200 nM at a flow rate of 10 μl/min.

Data were collected for 10 min and 1-min pulse of PBS, pH 8 containing0.05% Tween-20 was used to regenerate the surfaces.

Sensorgrams were generated and analyzed by using BIAevaluation softwareversion 3.1. The equilibrium RU observed for each injection was plottedagainst the concentration of Fc. The equilibrium Kd values were derivedby analysis of the plots by using the steady-state affinity modelincluded in the BIAevaluation software.

The Kd ratios thereby obtained confirmed that the S5A_41 is a betterbinder than Fc-H variant and S3A_07 is equivalent to Fc-H (Table 6).

IV.1.c. Summary of the Obtained Results

The table 6 hereunder shows FcRn binding characterization of variantsS5A_41, S3A_07 and Fc-H as compared to Fc_WT by (i) phage ELISA, (ii)ELISA and (iii) SPR. In all cases, variant S5A_41 displayed asignificantly higher capacity to bind FcRn than Fc_WT.

TABLE 6 FcRn binding characterisation of the Fc variants using ELISA andSurface Plasmon Resonance (SPR). For phage-ELISA and Fc-rec-ELISA, theratio refer to Variant specific signal divided by Fc-WT specific signal.For SPR, the ratio refers to Fc-WT Kd divided by Variant Kd. Fcphage-ELISA Fc-rec-ELISA SPR variants Ratio/Fc-WT Ratio/WT Ratio/Fc-WTFc-WT 1.0 1.0 1.0 Fc-H 3.2 27.1 7.6 S3A_07 3.5 25.3 5.2 S5A_41 9.0 66.310.7

IV.2. FcRn Binding Characterization of Other MS2 Variants as Compared toFc_WT by SPR and ELISA

Several Fc variants of MS2 were produced as described above in part III.The ability of each variant to bind FcRn was assayed by (i) SPR and by(ii) ELISA as described above in part IV.2.b and in part IV.2.c,respectively.

Several results are shown in FIG. 7.

Table 7 hereunder shows the results obtained for each MS2 variant testedby (i) SPR and (ii) ELISA. The previous results obtained by ELISA-phageare also indicated.

ELISA and SPR assays showed that all Fc variants are better binder thanwild-type Fc for FcRn, which correlated with the results previouslyobtained by ELISA assays on phage-Fc variants.

The Kd values of the MS2 variants at pH=6 ranged from 5.2 to 22.7 nM,which corresponds to an increase in affinity of 1.3 to 5.8 fold ascompared to Fc-WT.

TABLE 7 FcRn binding characterization of the Fc variants using ELISA andSurface Plasmon Resonance (SPR). For phage-ELISA, the ratio refers tovariant specific signal divided by Fc-WT specific signal. For ELISA, theratio refers to Fc-WT EC50 divided by variant EC50. For SPR, the ratiorefers to Fc-WT Kd divided by variant Kd. ELISA on phage- Fc-recombinantvariants SPR Name of ELISA ratio/ K_(d) (nM) at Ratio/ clone ratio/WTEC50 (nm) Fc-WT pH = 6 Fc-WT Fc-WT 1.0 461.2 1 30.2 1 Fc-H 3.2 16.6 2810.7 2.8 C6A_60 26.8 2.6 177 5.2 5.8 C6A_74 27.2 7.3 63 5.9 5.2 C6A_7827.8 4.7 97 5.8 5.2 C6A_69 28.4 3.7 124 7.2 4.2 T5A_74 27.6 5.5 85 7.14.2 C6A_66 24.6 4.9 95 9.7 3.1 C6A_72 25.7 7.8 59 12.4 2.4 T5A_78 25.64.7 97 12.4 2.4 55A_41 9.0 9.0 51 13.9 2.2 T5A_94 18.5 166.0 3 13.7 2.2T5A_58 26.1 9.8 47 15.4 2.0 T5A_81 14.9 144.8 3 22.7 1.3

The capacity of the Fc variants to bind FcRn at different pHs was alsoassessed by ELISA assay.

For each Fc variant previously tested, ELISA assays were performed at aconcentration providing an OD450 nm ranging from 0.8 and 1.0 whenperforming the ELISA assay at pH=6. The experimental conditions arethose described previously in part IV.1.a. Table 8 hereunder indicatesthe concentration of each Fc variant used for performing ELISA assays.

TABLE 8 Concentration of each Fc variant used to show the distinctbinding affinities to FcRn at different pHs. Fc Concentration (nM) Fc-WT200.0 Fc-H 6.2 C6A_69 1.2 T5A_74 1.2 C6A_60 1.2 S5A_41 2.0 C6A_78 2.5C6A_74 5.0 C6A_72 5.0 T5A_78 12.5 C6A_66 20.0 T5A_58 50.0

FIG. 7 shows the results of ELISA assays obtained for each variant.OD_(450 nm) correlates with the amount of Fc variants bound toimmobilized FcRn (detection of bound Fc variants with HRP anti-V5antibody). The higher the specific signal at OD_(450 nm) was, the higherthe binding of the Fc-variant to FcRn was.

FIG. 7 clearly shows that the binding of Fc variants with FcRn variesupon pH. As expected, the binding of Fc variants to FcRn at pH 7.4 isinsignificant as compared to the binding at pH 6.0.

It may be concluded that the amino acid modifications introduced toobtain Fc variants of the present invention may significantly increasethe binding to FcRn at pH 6.0 as compared to that of Fc-WT but may notsignificantly modify the binding at pH 7.4 which remains very low.

Example 2 Production of IgG Variants Based on Fc Variants and BiologicalCharacterization of Said IgG

I. Expression of the IgG Variants

I.1. Vector Construction

The Fc variants C6A_69; C6A_78; T5A_74; C6A_74; C6A_60 and C6-A66 wereprepared in a IgG format with an anti-CD20 specificity in YB2/0 cellline. For comparative purpose, IgG based on wild-type Fc (IgG-WT) wasalso produced.

In order to maximize productivity in the YB2/0 cell line, the fulllength heavy and light chains cDNA as well as Fc fragment coding thevariants were neo-synthesized with codon optimisation for Rattusnorvegicus. Unwanted features such as cryptic splicing sites orrestriction sites were removed. Only a restriction site (ApaI) waspresent at the junction variable/constant region.

In a first step, wild-type heavy chain was cloned between NheI and AscIin the expression vector CHK622-08, optimized for expression in YB2/0,resulting in the intermediate construct HCD20-Opti-GA. The optimizedlight chain was then cloned between SpeI and XbaI restriction sitesresulting in the final construct HKCD20-Opti-GA for expression of thewild-type anti-CD20 antibody (named IgG-WT hereunder).

Fc variants were prepared by replacing the wild IgG1 Fc fragment presentin HKCD20-Opti-GA by its appropriated version. This was cloned betweenApaI and AscI restriction sites (FIG. 8a).

Every fragment cloning was done by classical digestion/ligationprocedures, prior bacterial transformation. Expression constructs werescreened by enzymatic digestion plus PCR and validated by sequencing.

I.2. Cell Culture Production

5·10⁶ cells of the YB2/0 cell line (ATCC, CRL-1662) were electroporatedwith each expression linearised vector, then diluted at 25,000 cells/mLin RPMI 1640 medium+5% v/v dialysed FCS (InvitroGen) and dispensed under1 mL/well in 24-well plates. After 3 days of cell recovery, selectionpressure was applied by adding concentrated geneticin (Invitrogen) at0.5 g/L final and concentrated methotrexate (Sigma) at 25 mM final, 2mL/well. After 11 days of incubation, resistant cells were pooled foreach of the 8 constructs (encoding for the selected IgG MS2 variants,and IgG-WT) and progressively diluted with DMEM medium+5% v/v Ultra-lowIgG FCS (InvitroGen) until two (2) 2 L-roller bottles containing 0.9 Lof cell suspension each can be incubated at 2 rotation/minute. Cellswere allowed to grow and die (4 to 5 days) before supernatantcollection, clarification by low-speed centrifugation and volumereduction by ultra-filtration on Pellicon XL Filter (Millipore).

II. Purification and Characterisation of IgG Variants

The concentrated culture supernatants were injected into a HiTrapprotein A FF column (GE Healthcare). Bound antibodies were eluted withsodium citrate buffer 0.1 M, pH 3.0 and fractions were neutralized using100 μl of 1 M Tris pH 7.5 per ml of elution buffer. Fractions containingthe antibodies were pooled and dialyzed into PBS pH 6.0, and the sampleswere sterile-filtered (0.22 nm) and stored at 4° C.

The purified IgGs were characterised by SDS-PAGE under non-reducing andreducing conditions. Coomassie Blue-stained gels indicated that theIgGs, whatever the mutations, were purified to greater than 95%homogeneity and displayed the characteristic heavy and light chain bandsfor each IgG (FIG. 8b and FIG. 8c).

III. FcRn Binding Characterisation of the IgG Variants

The binding properties of IgG variants to FcRn were determined by threedistinct tests: (i) by ELISA assay, (ii) by SPR and (iii) by acompetition binding assay performed on Jurkat-cell line expressing atruncated FcRn in the presence of fluorescent-labelled Rituximab (ananti-CD20 IgG)

III.1. ELISA

III.1.a. Material and Method

The binding properties of the IgG variants produced in Y2B/O weredetermined using an ELISA test at pH6.0 with FcRn-p3 coated on wells.For comparative purpose, ELISA assay was also performed on IgG-WT

Purified IgG variants serially diluted in P6/5% skimmed milk/0.1%Tween-20 were tested on Maxisorp immunoplates previously coated with 0.1μg FcRn-p3/well and blocked with 5% skimmed milk in P6. After incubationfor 2 hours at 37° C., wells were washed 3 times with P6/0.1% Tween-20and bound IgG variants were detected with an HRP Fab′2 goat anti-humanFab′2 (Interchim).

For each IgG variants, the percentage of bound FcRn was plotted versusthe log of the concentration of IgG-variant. For each resulting bindingcurve, the measurement of the IgG concentration related to 50%saturation of the curve (EC50) was determined and compared to the EC50of WT-IgG.

III.1.b. ELISA Results

The ELISA tests showed that the produced IgG variants had an increasedbinding to FcRn as compared to that of WT-IgG. This fact is clearlyillustrated by binding curves (see FIG. 9) and by EC50 values.

As illustrated in table 9 hereunder, the EC50 of IgG-variants are atleast 5.8-fold lower than that of wild-type IgG. The best EC50 isobtained for C6A_69 variant.

TABLE 9 Concentration at 50% saturation (EC50) obtained from the ELISAbinding curve of each IgG variant. The ratio refers to WT EC50 dividedby variant EC50. IgG Variants EC50 (ng/ml) Ratio variant/WT WT 11060 1.0C6A_69 1021.6 10.8 C6A_78 1440.9 7.7 T5A_74 1191.8 9.3 C6A_74 2116.0 5.2C6A_60 1904.0 5.8 C6A_66 1900.4 5.8

111.2. IgG/FcRn Binding Affinity Measurements with SPR

III.2.a. Material and Method

The interaction of IgG-WT and IgG variants with recombinant, immobilizedhuman-FcRn was monitored by surface plasmon resonance (SPR) detectionusing a BIAcore X100 instrument (GE Healthcare). The experimentalprotocol was similar to that used for determining the affinity of Fcvariants (see paragraph IV.2.b. above).

The equilibrium RU observed for each injection was plotted against theconcentration of Fc. The equilibrium Kd values were derived by analysisof the plots by using the steady-state affinity model included in theBIAevaluation software. Kinetic parameters were determined by globalfitting of association and dissociation phase data with a model 1:2.

III.2.b. SPR Results

The binding affinity (Kd values) of the IgG-WT for human FcRn was 78.3nM. As illustrated in table 10, The Kd values of the 6 IgG variants wereranged from 10.5 to 18.8 nM which showed an increase in affinity forFcRn at pH 6.0 of 4 to 7 fold as compared to that of IgG-WT.

TABLE 10 Kd values obtained by SPR. The ratio refers to WT-Kd divided byvariant Kd. In order to determine the kinetic parameters, datasets forthe interaction of the IgG WT and variants with human FcRn were fit with1:2 model included in the BIAevaluation software. The curves obtainedwith IgG WT didn't fit with the 1:2 model whereas the curves obtainedwith the IgG variant C6A_66 and all other variants fit well with themodel 1:2 (data not shown). K_(d)(nM) Ratio WT/variant WT 78.27 1 C6A_6918.77 4 C6A_78 17.64 4 T5 A_74 10.55 7 C6 A_74 12.87 6 C6 A_60 13.79 6C6 A_66 15.18 5

As illustrated in table 11 hereunder, the enhanced affinity of the IgGvariants for human FcRn relative to the WT were predominantly driven byincreased association kinetics (kon values). Thus, the ratio that referto variants kon divided by WT-kon ranged from 13 to 23, indicating asignificant increase in affinity of variants to FcRn. The increasedvalues of Koff of the IgG variants relative to WT were ranged from 2 to4, displaying a faint impact of the dissociation as compared to theassociation.

TABLE 11 Rates of dissociation (koff) and association (kon) determinedby SPR k_(on) (×10⁵) k_(off) (1/s) K_(D) (nM) WT 0.36 0.00355 99 C6A_697.60 0.00837 11 C6A_78 8.19 0.00981 12 T5A_74 5.22 0.00885 17 C6A_745.81 0.01349 23 C6A_60 8.12 0.00788 9.7 C6A_66 4.83 0.01264 26

III.3. Binding to Jurkat-FcRn

III.3.a. Material and Method

Competition immunofluorescence assays were performed to evaluate theability of IgG WT and variants to interact with FcRn by a method adaptedfrom that described by Dall'Ozzo et al. (Dall'Ozzo S, Tartas S, PaintaudG, Cartron G, Colombat P, Bardos P, Watier H, Thibault G, Cancer Res.,2004 Jul. 1; 64(13):4664-9).

Briefly, IgG WT and variants were diluted in PBS pH6 at a finalconcentration ranging from 0.06 to 2 mg/ml and incubated with JurkatFcRn (150000 cell) in the presence of Alexa-conjugated Rituximab(labelled Rituximab) at a concentration of 50 μg/ml. After 20 minutes,the cells were analyzed by flow cytometry in order to quantifyAlexa-Rituximab binding. The results were expressed as a percentage ofthe mean fluorescence intensity (MFI), 100% refers to the meanfluorescence intensity (MFI) obtained with Alexa-conjugated Rituximabalone (i.e. without competitor) and 0% refers to the MFI value measuredwhen Jurkat FcRn was not incubated with Alexa conjugated Rituximab. Eachexperiment was done in triplicate.

Controls comprise the incubation of (i) unlabelled Rituximab or (ii)IgG-WT.

For each tested IgG, the MFI was plotted versus the log of IgGconcentration. The concentration (IC50) of each tested IgG whichprovides an inhibition of 50% of the MFI signal was determined.

A general description of this assay may also be found in the Frenchpatent application published as FR 2 894 983.

III.3.b. Experimental Results

Several results are shown in FIG. 11 where the binding or Ritixan and ofvarious variants according to the invention to Jurkat FcRn has beendetermined as described in the Materials and Methods Section above andexpressed as mean fluorescence intensity (MFI) values.

As illustrated in table 12 hereunder, the IC50 obtained for the variantsof the invention are significantly lower than that obtained for WT-IgG.The decrease in IC50 for IgG variants of the invention was from 40 to60-fold except for C6A_66.

TABLE 12 IC50 obtained for binding competition assay performed on Jurkatcells expressing FcRn in the presence of fluorescent-labelled Rituximab.The “50% RTX = 1” values consist of IC50 values that are also expressedμg/ml. IC50 (μg/ml) 50% RTX = 1 Rituximab NA ≠1 WT 219 5 C6A_69 4 240C6A_78 4 275 T5A_74 4 224 C6A_74 5 200 C6A_60 3 234 C6A_66 21 48

III.4. Conclusion

The three distinct tests performed to characterize the bindingproperties of IgG variants to FcRn provided consistent results. In allcase, IgG variants of the invention displayed a significant increasedbinding to FcRn as compared to that of IgG wild type.

IV. Functional Characterisation IgG Variants and Comparison with IgG-WTand LFB-R603

The ability of IgG variants to bind Fcγ receptors and their ADCC and CDCactivities were assessed in order to fully-characterize their biologicalfunctions.

IV.1. Binding of I IgG Variants Binding to hFcγRIIIA

IV.1.a. ELISA Assay: Binding of IgG Variant to Immobilized RecombinanthFcγRIIIA

The human recombinant FcγRIIIA (F158 allotype) was biotinylated withEZ-link NHS-PEO kit (Pierce), diluted at 1 μg/ml in assay buffer (Tris25 mM, NaCl 150 mM, pH 7.35, 0.05% Tween-20, 0.1% BSA) and coated ontoReact-Bind™ streptavidin ELISA plates (Pierce) for 2 h at roomtemperature. During this incubation time, IgG-F(ab′)2 anti-F(ab′)2complexes were prepared in assay buffer by mixing 5 μg/ml of IgG and 2μg/ml F(ab′)2 anti-human F(ab′)2 labelled with horseradish peroxidase(Jackson ImmunoResearch) for 2 h at room temperature. Serial dilutionsof complexes were added to plates and incubated for 2 h at roomtemperature under gentle shaking. After washing plates with assaybuffer, bound complexes to hFcγRIIIA were detected with TMB (Pierce).Absorbance at 450 nm was read using a plate reader (Tecan).

For each IgG variants, the percentage of bound FcγRIIIA (which isobtained from OD450 nm) was plotted versus the concentration ofIgG-variant.

As shown in FIG. 10, the binding of IgG variants to hFcγRIIIA is similarto that of the IgG WT, except for variant C6A_66 which fails to bindhFcγRIIIA.

IV.2. ADCC Activity

The natural killer (NK cells) cells were purified from the peripheralblood of healthy donors by the negative depletion technique developed bythe company Miltenyi. The ADCC test comprises incubating the NK cellswith the target cells of the Raji line that express the CD20 receptor,in the presence of different concentrations of anti-CD20 antibodies.After 16 hours of incubation, the cytotoxicity induced by the anti-CD20antibodies is chromogenically measured by quantifying in cellsupernatants the level of an intracellular enzyme called lactatedehydrogenase (LDH) which is released by the lysed target cells. Theresults are shown in FIG. 12.

The specific lysis results are expressed as the percent lysis as afunction of antibody concentration. EC50 (quantity of antibody thatinduces 50% of maximum lysis) were calculated using PRISM software.Control experiments were performed with (i) Rituximab, (ii) WT-IgGproduced in Y2/0 cells and LFB-R603 which is an anti-CD20 antibody knownto have ADDC function that has been described by de Romeuf et al. in2004 (de Romeuf C, Dutertre C A, Le Garff-Tavernier M, Fournier N,Gaucher C, Glacet A, Jorieux S, Bihoreau N, Behrens C K, Béliard R,Vieillard V, Cazin B, Bourel D, Prost J F, Teillaud J L, Merle-Béral H.Chronic lymphocytic leukaemia cells are efficiently killed by ananti-CD20 monoclonal antibody selected for improved engagement ofFcgammaRIIIA/CD16. Br J Haematol. 2008 March; 140(6):635-43). as well asin the PCT application no WO 2006/064121.

Table 13 hereunder shows the EC50 for each variant and compares the ADCCfunction of IgG variants with that of LFB-R603 and WT-IgG.

All IgG variants display ADCC activity except C6A_66 variant. Thisvariant has no ADCC activity which is consistent with its very lowaffinity for FcγRIII.

It should be noticed that C6A_69, C6A_60 and C6A_74 have an increasedADCC activity as compared to IgG-WT. The other variants (namely C6A_78and T5A_75) have an ADCC activity similar to that of IgG-WT.

TABLE 13 EC50 (quantity of antibody that induces 50% of maximum lysis)obtained from ADCC assay. The ratio refers variant EC50 divided byLFB-R603 EC50. EC50 (μg/ml) Ratio R603/Variant LFB-R603 0.2 1.0Rituximab >5000 N.A. WT 1.2 6.0 C6A_69 0.5 2.3 C6A_78 1.0 4.7 T5A_74 0.73.3 C6A_74 0.2 0.9 C6A_60 0.3 1.6 C6A_66 >5000 N.A.

IV.3. CDC Activity

In this technique, the target CD20+ cells of the Raji line wereincubated with different concentrations of anti-CD20 antibodies (0 to5000 ng/ml) in the presence of baby rabbit serum as a source ofcomplement (Cedarlane ref.: CL3441, dilution to 1/10). After 1 hour ofincubation at 37° C., the quantity of LDH released in the supernatant bythe lysed target cells is measured chromogenically (Roche AppliedSciences Cytotoxicity Detection Kit) and is used to quantity thecomplement-dependent cytotoxicity mediated by the antibodies. Theresults are expressed as a percentage of lysis. EC50 (quantity ofantibody that induces 50% of maximum lysis) and Emax (percentage ofmaximum lysis) were calculated using PRISM software.

Table 14 hereunder shows the Emax and EC50 obtained for each variant.

The level of CDC activity varies upon IgG variants.

C6A_78 and C6A_60 have a CDC activity significantly higher that ofIgG-WT whereas C6A_69, T5_74 and C6A_66 display low CDC activity.

The CDC activity of C6A_74 variant is similar to that of IgG-WT.

TABLE 14 EC50 (quantity of antibody that induces 50% of maximum lysis)obtained from CDC assay. Emax (lysis %) EC50 (ng/ml) LFB-R603 61.87514.0 Rituximab 65.60 419.0 WT 57.32 541.1 C6A_69 N.A. >5000 C6A_7875.99 117.3 T5A_74 N.A. >5000 C6A_74 59.90 458.4 C6A_60 77.22 92.66C6A_66 10.28 935.1

IV.3. Conclusion

The six IgG variants of the invention recombinantly produced in Y2B/0cell line have an increased binding to FcRn receptor as compared to theIgG-WT (produced in the same host cell and in the same condition).

IgG variants of the invention have at least the same binding affinity toFcgRIII and the at least the same ADCC activity than IgG-WT, exceptC6AA_66 which shows poor affinity for FcgRIII.

The IgG variants display distinct CDC activities.

To conclude, in some aspects, amino acid modifications according to theinvention enable to obtain IgG variants which have an increased bindingfor FcRn combined with one or more Fc effector activities which are atleast similar to that of the corresponding parent IgG (i.e IgG-WT).

In other aspect, amino acid modifications according to the inventionenable to obtain IgG variants which have an increased binding for FcRncombined with at least one decreased Fc effector activity such as CDC orADCC.

Table 15 hereunder shows the main conclusions concerning IgG variants ofthe present study.

TABLE 15 Main results obtained for the IgG variants of the invention ascompared to IgG-WT; Liaison Variant Mutations FcRn ADCC CDC C6A_69T307A/N315D/A330V/ ++

E382V/N389T/N434Y C6A_78 T256N/A378V/S383N/N434Y ++ ✓

T5A_74 N315D/A330V/N361D/A378V/ ++ ✓

N434Y C6A_74 V259I/N315D/N434Y ++

✓ C6A_60 P230S/N315D/M428L/N434Y ++

C6A_66 E294del/T307P/N434Y ++

++: Increased binding to FcRn as compared to WT-IgG;

: Increased activity as compared to WT-IgG;

: Decreased activity as compared to WT-IgG; ✓: Activity similar to thatof WT-IgG

TABLE 7 Sequences included in the sequence listing SEQ ID NO: Sequences1 Human IgG1 Fc (residues 226-447 according to EU index as Kabat) 2Human IgG2 Fc 3 Human IgG3 Fc 4 Human IgG4 Fc 5 Primer 6 Primer 7 Primer8 Primer 9 Primer 10 Primer 11 Primer 12 Fragment of heavy chain ofhuman IgG1 Glm1,17 allotype 13 Fragment of heavy chain of human IgG1Glm3 allotype 14 Fragment of the heavy chain of human IgG2 15 Fragmentof the heavy chain of human IgG3 16 Fragment of the heavy chain of humanIgG4

The invention claimed is:
 1. A variant of a parent polypeptidecomprising a Fc region, which variant exhibits increased binding to FcRnas compared to said parent polypeptide and comprises a combination ofamino acid modifications selected from the group consisting of:N315D/A330V/N361D/A378V/N434Y, V264E/N315D/A378V/N390S/G420R/N434Y,N315D/A378V/N434Y, N315D/A330V/A378V/N434Y, N315D/K334E/A378V/N434Y,V264E/N315D/A378V, P228R/N315D/A330V/N361D/A378V/N434Y,P228R/P230S/N315D/A330V/N361D/A378V/N434Y,P228L/N315D/A330V/N361D/A378V/N434Y,P228L/P230S/N315D/A330V/N361D/A378V/N434Y,P230S/N315D/A330V/N361D/A378V/N434Y, P230T/V264E/N315D/K370R/A378V, ofthe Fc region, wherein the numbering of the amino acids in the Fc regionis that of the EU index as in Kabat.
 2. The variant according to claim1, wherein said variant is an antibody.
 3. The variant according toclaim 2, wherein said antibody is an IgG antibody.
 4. A pharmaceuticalcomposition comprising a variant as defined in claim
 1. 5. A medicamentcomprising a variant according to claim
 1. 6. An isolated nucleic acidencoding a variant as defined in claim
 1. 7. A vector comprising thenucleic acid of claim
 6. 8. An isolated host cell containing the vectorof claim
 7. 9. A method for producing a variant according to claim 1,comprising culturing a host cell containing a vector comprising anisolated nucleic acid encoding said variant so that the nucleic acid isexpressed.
 10. A variant according to claim 1, wherein the Fc region ofthe parent polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, orSEQ ID NO: 4.